Anti-egfr antibodies and antibody drug conjugates

ABSTRACT

The invention relates to anti-epidermal growth factor (EGFR) antibodies and antibody drug conjugates (ADCs), including compositions and methods of using said antibodies and ADCs.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/349,692, filed on Nov. 11, 2016, which is a divisional of U.S.patent application Ser. No. 14/664,453, filed on Mar. 20, 2015, now U.S.Pat. No. 9,493,568, issued Nov. 15, 2016, which in turn claims priorityto U.S. Provisional Patent Application Ser. No. 61/968,819, filed onMar. 21, 2014, the entire contents of which are incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 9, 2017, isnamed A103017_1020USD2_SL.txt and is 110,592 bytes in size.

BACKGROUND OF THE INVENTION

The human epidermal growth factor receptor (also known as HER-1 orErb-B1, and referred to herein as “EGFR”) is a 170 kDa transmembranereceptor encoded by the c-erbB protooncogene, and exhibits intrinsictyrosine kinase activity (Modjtahedi et al., Br. J. Cancer 73:228-235(1996); Herbst and Shin, Cancer 94:1593-1611 (2002)). SwissProt databaseentry P00533 provides the sequence of human EGFR. EGFR regulatesnumerous cellular processes via tyrosine-kinase mediated signaltransduction pathways, including, but not limited to, activation ofsignal transduction pathways that control cell proliferation,differentiation, cell survival, apoptosis, angiogenesis, mitogenesis,and metastasis (Atalay et al., Ann. Oncology 14:1346-1363 (2003); Tsaoand Herbst, Signal 4:4-9 (2003); Herbst and Shin, Cancer 94:1593-1611(2002); Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)).

Known ligands of EGFR include EGF, TGFA/TGF-alpha, amphiregulin,epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-bindingEGF. Ligand binding by EGFR triggers receptor homo- and/orheterodimerization and autophosphorylation of key cytoplasmic residues.The phosphorylated EGFR recruits adapter proteins like GRB2 which inturn activate complex downstream signaling cascades, including at leastthe following major downstream signaling cascades: the RAS-RAF-MEK-ERK,PI3 kinase-AKT, PLCgamma-PKC, and STATs modules. Thisautophosphorylation also elicits downstream activation and signaling byseveral other proteins that associate with the phosphorylated tyrosinesthrough their own phosphotyrosine-binding SH2 domains. These downstreamsignaling proteins initiate several signal transduction cascades,principally the MAPK, Akt and JNK pathways, leading to cellproliferation. Ligand binding by EGFR may also activate the NF-kappa-Bsignaling cascade. Ligand binding also directly phosphorylates otherproteins like RGS16, activating its GTPase activity and potentiallycoupling the EGF receptor signaling to G protein-coupled receptorsignaling. Ligand binding also phosphorylates MUC1 and increases itsinteraction with SRC and CTNNB1/beta-catenin.

Overexpression of EGFR has been reported in numerous human malignantconditions, including cancers of the bladder, brain, head and neck,pancreas, lung, breast, ovary, colon, prostate, and kidney. (Atalay etal., Ann. Oncology 14:1346-1363 (2003); Herbst and Shin, Cancer94:1593-1611 (2002); and Modjtahedi et al., Br. J. Cancer 73:228-235(1996)). In many of these conditions, the overexpression of EGFRcorrelates or is associated with poor prognosis of the patients. (Herbstand Shin, Cancer 94:1593-1611 (2002); and Modjtahedi et al., Br. J.Cancer 73:228-235 (1996)). EGFR is also expressed in the cells of normaltissues, particularly the epithelial tissues of the skin, liver, andgastrointestinal tract, although at generally lower levels than inmalignant cells (Herbst and Shin, Cancer 94:1593-1611 (2002)).

A significant proportion of tumors containing amplifications of the EGFRgene (i.e., multiple copies of the EGFR gene) also co-express atruncated version of the receptor (Wikstrand et al. (1998) J.Neurovirol. 4, 148-158) known as de2-7 EGFR, ΔEGFR, EGFRvIII, or Δ2-7(terms used interchangeably herein) (Olapade-Olaopa et al. (2000) Br. J.Cancer. 82, 186-94). The rearrangement seen in the de2-7 EGFR results inan in-frame mature mRNA lacking 801 nucleotides spanning exons 2-7 (Wonget al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2965-9; Yamazaki et al.(1990) Jpn. J. Cancer Res. 81, 773-9; Yamazaki et al. (1988) Mol. Cell.Biol. 8, 1816-20; and Sugawa et al. (1990) Proc. Natl. Acad. Sci. U.S.A.87, 8602-6). The corresponding EGFR protein has a 267 amino aciddeletion comprising residues 6-273 of the extracellular domain and anovel glycine residue at the fusion junction (Sugawa et al., 1990). Thisdeletion, together with the insertion of a glycine residue, produces aunique junctional peptide at the deletion interface (Sugawa et al.,1990).

EGFRvIII has been reported in a number of tumor types including glioma,breast, lung, ovarian and prostate (Wikstrand et al. (1997) Cancer Res.57, 4130-40; Olapade-Olaopa et al. (2000) Br. J. Cancer. 82, 186-94;Wikstrand, et al. (1995) Cancer Res. 55, 3140-8; Garcia de Palazzo etal. (1993) Cancer Res. 53, 3217-20). While this truncated receptor doesnot bind ligand, it possesses low constitutive activity and imparts asignificant growth advantage to glioma cells grown as tumor xenograftsin nude mice (Nishikawa et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91,7727-31) and is able to transform NIH3T3 cells (Batra et al. (1995) CellGrowth Differ. 6, 1251-9) and MCF-7 cells. The cellular mechanismsutilized by the de2-7 EGFR in glioma cells are not fully defined but arereported to include a decrease in apoptosis (Nagane et al. (1996) CancerRes. 56, 5079-86) and a small enhancement of proliferation (Nagane etal., 1996). As expression of this truncated receptor is restricted totumor cells it represents a highly specific target for antibody therapy.

Antibody drug conjugates (ADC) represent a new class of therapeuticscomprising an antibody conjugated to a cytotoxic drug via a chemicallinker. The therapeutic concept of ADCs is to combine bindingcapabilities of an antibody with a drug, where the antibody is used todeliver the drug to a tumor cell by means of binding to a target surfaceantigen.

Accordingly, there remains a need in the art for anti-EGFR antibodiesand ADCs that can be used for therapeutic purposes in the treatment ofcancer.

SUMMARY OF THE INVENTION

In certain aspects, the present invention provides for anti-EGFRantibodies and antibody drug conjugates (ADCs) that specifically bind toEGFRvIII.

In one embodiment, the invention features anti-human epidermal growthfactor receptor (anti-hEGFR) antibodies, or antigen binding portionsthereof, that bind to an epitope within the amino acid sequenceCGADSYEMEEDGVRKC (SEQ ID NO: 45) or competes with a second anti-hEGFRantibody for binding to epidermal growth factor receptor variant III(EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay, wherein thesecond anti-EGFR antibody comprises a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 1 and a lightchain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 5; binds to EGFR(1-525) (SEQ ID NO: 47) with a dissociationconstant (K_(d)) of about 1×10⁻⁶ M or less, as determined by surfaceplasmon resonance; and inhibits tumor growth in an in vivo humannon-small-cell lung carcinoma (NSCLC) xenograft assay with a tumorgrowth inhibition % (TGI %) of at least about 50% relative to a humanIgG antibody which is not specific for EGFR, wherein the human IgGantibody is administered in the NSCLC xenograft assay at the same doseand frequency as the anti-hEGFR antibody, or antigen binding portionthereof.

In certain embodiments of the invention, the antibodies, or antigenbinding portions thereof, bind to EGFR (1-525) (SEQ ID NO: 47) with aK_(d) of between about 1×10⁻⁶ M and about 1×10⁻¹⁰ M, as determined bysurface plasmon resonance.

In other embodiments of the invention, the antibodies, or antigenbinding portions thereof, bind to EGFR (1-525) (SEQ ID NO: 47) with aK_(d) of between about 1×10⁻⁶ M and about 1×10⁻⁷ M, as determined bysurface plasmon resonance.

In certain embodiments, the antibodies, or antigen binding portionsthereof, of the invention bind to EGFRvIII (SEQ ID NO: 33) with a K_(d)of about 8.2×10⁻⁹ M or less, as determined by surface plasmon resonance.In further embodiments, the antibody, or antigen binding portionthereof, binds to EGFRvIII (SEQ ID NO: 33) with a K_(d) of between about8.2×10⁻⁹ M and about 6.3×10⁻¹⁰ M, as determined by surface plasmonresonance. In some embodiments, the antibody, or antigen binding portionthereof, binds to EGFRvIII (SEQ ID NO: 33) with a K_(d) of between about8.2×10⁻⁹ M and about 2.0×10⁻⁹ M, as determined by surface plasmonresonance.

In yet other embodiments of the invention, the antibodies, or antigenbinding portions thereof, inhibit tumor growth by at least about 60% inan in vivo human non-small-cell lung carcinoma (NSCLC) xenograft assayrelative to a human IgG antibody which is not specific for EGFR.

In certain embodiments, the invention features antibodies, or antigenbinding portions thereof, that inhibits tumor growth by at least about70% in an in vivo human non-small-cell lung carcinoma (NSCLC) xenograftassay relative to a human IgG antibody which is not specific for EGFR.In certain embodiments, the antibodies, or antigen binding portionsthereof, inhibit tumor growth by at least about 80% in an in vivo humannon-small-cell lung carcinoma (NSCLC) xenograft assay relative to ahuman IgG antibody which is not specific for EGFR.

In some embodiments, the antibodies, or antigen binding portionsthereof, comprise a heavy chain CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 12, a heavy chain CDR2 domaincomprising the amino acid sequence set forth in SEQ ID NO: 11, and aheavy chain CDR1 domain comprising the amino acid sequence set forth inSEQ ID NO: 10; and a light chain CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 8, a light chain CDR2 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 7, and a light chainCDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:6. In yet another embodiment, the antibodies, or antigen bindingportions thereof, comprise a heavy chain variable region comprising theamino acid sequence set forth in SEQ ID NO: 9, and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 5. In other embodiments, the antibodies, or antigen binding portionsthereof, comprise a heavy chain constant region comprising the aminoacid sequence set forth in SEQ ID NO: 41 and/or a light chain constantregion comprising the amino acid sequence set forth in SEQ ID NO: 43. Ina further embodiment, the antibodies, or antigen binding portionsthereof, comprise a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 15, and a light chain comprising the amino acidsequence set forth in SEQ ID NO: 13. In another embodiment, the theantibodies, or antigen binding portions thereof, are conjugated to anauristatin.

The invention also provides, in certain embodiments, isolated nucleicacids encoding an antibodies, or antigen binding portions thereof, likethat described herein.

The invention also includes, in certain embodiments, anti-hEGFRantibodies, or antigen binding portions thereof, comprising a lightchain CDR3 domain comprising the amino acid sequence set forth in SEQ IDNO: 40, a light chain CDR2 domain comprising the amino acid sequence setforth in SEQ ID NO: 39, and a light chain CDR1 domain comprising theamino acid sequence set forth in SEQ ID NO: 38; and a heavy chain CDR3domain comprising the amino acid sequence set forth in SEQ ID NO: 37, aheavy chain CDR2 domain comprising the amino acid sequence set forth inSEQ ID NO: 36, and a heavy chain CDR1 domain comprising the amino acidsequence set forth in SEQ ID NO: 35.

In certain embodiments, the invention features anti-hEGFR antibodies, orantigen binding portions thereof, comprising a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,and 78; and a light chain variable region comprising an amino acidsequence selected from the group consisting of 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, and 79.

In other embodiments, the invention includes anti-hEGFR antibodies, orantigen binding portions thereof, comprising a heavy chain CDR set(CDR1, CDR2, and CDR3) selected from the group consisting of SEQ ID NOs:10, 11, and 12; SEQ ID NOs: 16, 17, and 18; SEQ ID NOs: 10, 11, and 19;SEQ ID NOs: 20, 11, and 12; SEQ ID NOs: 21, 3, and 22; SEQ ID NOs: 16,17, and 19; SEQ ID NOs: 2, 3, and 4; SEQ ID NOs: 10, 3, and 12; SEQ IDNOs: 80, 11, and 18; SEQ ID NOs: 80, 3, and 18; SEQ ID NOs: 20, 3, and12; SEQ ID NOs: 80, 11, and 12; and SEQ ID NOs: 81, 11, and 22; and alight chain CDR set (CDR1, CDR2, and CDR3) selected from the groupconsisting of SEQ ID NOs: 6, 7, and 8; SEQ ID NOs: 23, 24, and 25; SEQID NOs: 26, 27, and 28; SEQ ID NOs: 29, 30, and 31; SEQ ID NOs: 6, 7,and 84; SEQ ID NOs: 82, 83, and 31; and SEQ ID NOs: 82, 27, and 85,wherein the antibodies, or antigen binding portions thereof, does notcomprise both the heavy chain CDR set of SEQ ID NOs: 2, 3, and 4, andthe light chain CDR set of SEQ ID NOs: 6, 7, and 8. In some embodiments,the antibodies, or antigen binding portions thereof, comprises a heavychain constant region comprising the amino acid sequence set forth inSEQ ID NO: 41 and/or a light chain constant region comprising the aminoacid sequence set forth in SEQ ID NO: 43.

In some embodiments of the invention, the antibodies, or antigen bindingportions thereof, comprise a heavy chain immunoglobulin constant domainselected from the group consisting of a human IgG constant domain, ahuman IgM constant domain, a human IgE constant domain, and a human IgAconstant domain. In some embodiments, the IgG constant domain isselected from the group consisting of an IgG1 constant domain, an IgG2constant domain, an IgG3 constant domain, and an IgG4 constant domain.In other embodiments, the antibody is a multispecific antibody.

In other embodiments of the invention, the antibodies, or antigenbinding portions thereof, comprise a Fab, a Fab′, a F(ab′)2, a Fv, adisulfide linked Fv, an scFv, a single domain antibody, and a diabody.

In yet other embodiments of the invention, the antibodies, or antigenbinding portions thereof, are conjugated to an imaging agent. In certainembodiments of the invention, the imaging agent is selected from thegroup consisting of a radiolabel, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, and biotin.In other embodiments of the invention, the radiolabel is indium. In yetother embodiments, the invention includes a pharmaceutical compositioncomprising the antibody, or antigen binding portion thereof, and apharmaceutically acceptable carrier.

The invention also includes, in some embodiments, an antibody drugconjugate (ADC) comprising the antibody, or antigen binding portionthereof, described herein, conjugated to at least one drug. In certainembodiments, the antibody is an anti-human epidermal growth factorreceptor (anti-hEGFR) antibody, or antigen binding portion thereof, thatbinds to an epitope within the amino acid sequence CGADSYEMEEDGVRKC (SEQID NO: 45) or competes with a second anti-hEGFR antibody for binding toepidermal growth factor receptor variant III (EGFRvIII) (SEQ ID NO: 33)in a competitive binding assay, wherein the second anti-EGFR antibodycomprises a heavy chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 1 and a light chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 5; binds toEGFR(1-525) (SEQ ID NO: 47) with a dissociation constant (K_(a)) ofabout 1×10⁻⁶ M or less, as determined by surface plasmon resonance; andinhibits tumor growth in an in vivo human non-small-cell lung carcinoma(NSCLC) xenograft assay with a tumor growth inhibition % (TGI %) of atleast about 50% relative to a human IgG antibody which is not specificfor EGFR, wherein the human IgG antibody is administered in the NSCLCxenograft assay at the same dose and frequency as the anti-hEGFRantibody, or antigen binding portion thereof. In one embodiment of theinvention, the at least one drug is selected from the group consistingof an anti-apoptotic agent, a mitotic inhibitor, an anti-tumorantibiotic, an immunomodulating agent, a nucleic acid for gene therapy,an alkylating agent, an anti-angiogenic agent, an anti-metabolite, aboron-containing agent, a chemoprotective agent, a hormone agent, ananti-hormone agent, a corticosteroid, a photoactive therapeutic agent,an oligonucleotide, a radionuclide agent, a radiosensitizer, atopoisomerase inhibitor, and a tyrosine kinase inhibitor. In certainembodiments, the mitotic inhibitor is a dolastatin, an auristatin, amaytansinoid, and a plant alkaloid. In certain embodiments, the drug isa dolastatin, an auristatin, a maytansinoid, and a plant alkaloid. Anexample of an auristatin is monomethylaurisatin F (MMAF) ormonomethyauristatin E (MMAE). Examples of maytansinoids include, but arenot limited to, DM1, DM2, DM3, and DM4. In certain embodiments, theanti-tumor antibiotic is selected from the group consisting of anactinomycine, an anthracycline, a calicheamicin, and a duocarmycin. Incertain embodiments, the actinomycine is a pyrrolobenzodiazepine (PBD).

The invention also includes, in some embodiments, an ADC comprising ananti-EGFR antibody conjugated to an auristatin, wherein the antibodycomprises a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 10; and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In one embodiment, the antibody comprises aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 9, and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 5. In yet another embodiment, the inventionincludes antibodies, or antigen binding portions thereof comprising aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain comprising the amino acid sequence of SEQ ID NO:13.

The invention also includes, in some embodiments, an ADC comprising ananti-EGFR antibody conjugated to at least one drug (including, but notlimited to, MMAE), wherein between 1 to 8 molecules of the drug areconjugated to the antibody. In one embodiment, 1 to 4 molecules of thedrug are conjugated to the antibody of the ADC. In one embodiment, 2 to4 molecules of the drug are conjugated to the antibody of the ADC.

The invention also includes, in some embodiments, an ADC comprising ananti-EGFR antibody conjugated to at least one drug, wherein the drug isconjugated via a maleimidocaproyl, valine-citrulline linker. In afurther embodiment, the drug is conjugated to the antibody via amaleimidocaproyl, valine-citrulline, p-aminobenzyloxycarbamyl (PABA)linker.

The invention also includes, in some embodiments, an ADC comprising ananti-EGFR IgG1 antibody covalently linked to monomethylauristatin E(MMAE) via a linker (e.g., maleimidocaproyl, valine-citrulline). Incertain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 9, andcomprises a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO: 5. In certain embodiments, 1 to 4molecules of MMAE are linked to the antibody.

The invention also includes, in some embodiments, an ADC comprising ananti-EGFR IgG1 antibody covalently linked to maleimidocaproyl,valine-citrulline, p-aminobenzyloxycarbamyl-monomethylauristatin E(mc-vc-PABA-MMAE), wherein the antibody comprises a heavy chain variableregion comprising the amino acid sequence set forth in SEQ ID NO: 9, andcomprises a light chain variable region comprising the amino acidsequence set forth in SEQ ID NO: 5, and wherein 1 to 4 molecules of MMAEare linked to the antibody. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 15, and comprises a light chain comprising the amino acidsequence set forth in SEQ ID NO: 13. In certain embodiments, 2 to 4molecules of MMAE are linked to the antibody. In certain embodiments,the EGFR antibody is linked to mc-vc-PABA-MMAE as depicted in FIG. 11.

The invention also includes, in some embodiments, an EGFR-directed ADCcomprising an IgG1 antibody specific for human EGFR, MMAE, and a linkerthat covalently attaches MMAE to the antibody. In certain embodiments,the antibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 10; andcomprises a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 6. In one embodiment,the antibody comprises a heavy chain variable region comprising theamino acid sequence set forth in SEQ ID NO: 9, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 5. Inyet another embodiment, the invention includes antibodies, or antigenbinding portions thereof comprising a heavy chain comprising the aminoacid sequence set forth in SEQ ID NO: 15, and a light chain comprisingthe amino acid sequence of SEQ ID NO: 13.

In yet other embodiments, the invention includes a pharmaceuticalcomposition comprising an ADC mixture comprising a plurality of the ADCdescribed herein, and a pharmaceutically acceptable carrier. In certainembodiments, the ADC mixture has an average drug to antibody ratio (DAR)of 2 to 4. In other embodiments the ADC mixture comprises ADCs eachhaving a DAR of 2 to 8. In certain embodiments, the ADC mixture has anaverage drug to antibody (DAR) of about 2.4 to about 3.6.

In certain embodiments, the invention includes methods for treating asubject having cancer, comprising administering the pharmaceuticalcomposition described herein to the subject, such that the subjecthaving cancer is treated. In one embodiment, the cancer is selected fromthe group consisting of breast cancer, lung cancer, a glioblastoma,prostate cancer, pancreatic cancer, colon cancer, head and neck cancer,and kidney cancer. In one embodiment, the cancer is selected from thegroup consisting of breast cancer, lung cancer, a glioblastoma, prostatecancer, pancreatic cancer, colon cancer, colorectal cancer, head andneck cancer, mesothelioma, kidney cancer, squamous cell carcinoma,triple negative breast cancer, and non-small cell lung cancer. In oneembodiment, the cancer is breast cancer. In one embodiment, the canceris lung cancer. In one embodiment, the cancer is prostate cancer. In oneembodiment, the cancer is pancreatic cancer. In one embodiment, thecancer is colon cancer. In one embodiment, the cancer is head and neckcancer. In one embodiment, the cancer is kidney cancer. In oneembodiment, the cancer is colorectal cancer. In one embodiment, thecancer is mesothelioma. In one embodiment, the cancer is squamous cellcarcinoma. In one embodiment, the cancer is triple negative breastcancer. In one embodiment, the cancer is non-small cell lung cancer. Incertain embodiments, the squamous cell carcinoma is squamous lung canceror squamous head and neck cancer.

In yet another embodiment, the cancer contains amplifications of EGFR oroverexpresses EGFR. In certain embodiments, the cancer is characterizedas having EGFR overexpression. In certain embodiments, the cancer ischaracterized as having EGFR amplification.

The invention further includes, in certain embodiments, methods forinhibiting or decreasing solid tumor growth in a subject having a solidtumor, comprising administering the pharmaceutical composition describedherein to the subject having the solid tumor, such that the solid tumorgrowth is inhibited or decreased. In certain embodiments, the solidtumor is characterized as having EGFR overexpression. In certainembodiments, the solid tumor is characterized as having EGFRamplification.

In one embodiment of the invention, the invention provides for methodsfor inhibiting or decreasing solid tumor growth in a subject having asolid tumor, comprising administering to the subject having the solidtumor an effective amount of the antibody or ADC described herein, suchthat the solid tumor growth is inhibited or decreased.

In certain embodiments, the solid tumor is an EGFR expressing solidtumor or an EGFRvIII positive solid tumor. In other embodiments, thesolid tumor is a non-small cell lung carcinoma or a glioblastoma. Inother embodiments, the solid tumor is a squamous cell carcinoma.

In one embodiment of the invention, the invention provides for a methodfor treating a subject having cancer, comprising administering aneffective amount of an ADC comprising an anti-EGFR antibody, or antigenbinding portion thereof, conjugated to at least one auristatin, whereinthe anti-EGFR antibody, or antigen binding portion thereof, is an IgGisotype; comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence set forth in SEQ ID NO: 12, aCDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:11, and a CDR1 domain comprising the amino acid sequence set forth inSEQ ID NO: 10; and comprises a light chain variable region comprising aCDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:8, a CDR2 domain comprising the amino acid sequence set forth in SEQ IDNO: 7, and a CDR1 domain comprising the amino acid sequence set forth inSEQ ID NO: 6. In certain embodiments, the antibody, or antigen bindingportion thereof, is linked to mc-vc-PABA-MMAE.

In certain embodiments, the invention includes methods for treating asubject having cancer, comprising administering the pharmaceuticalcomposition described herein to the subject in combination with anadditional agent or additional therapy. In certain embodiments, theadditional agent is selected from the group consisting of an anti-PD1antibody (e.g., pembrolizumab (Keytruda®) or nivolumab), an anti-CTLA-4antibody (e.g., ipilimumab), ibrutinib, duvelisib, idelalisib,venetoclax, and temozolomide. In certain embodiments, the additionaltherapy is radiation. In certain embodiments, the additional agent is ananti-PD1 antibody (e.g., pembrolizumab (Keytruda®) or nivolumab). Incertain embodiments, the additional agent is an anti-CTLA-4 antibody(e.g., ipilimumab). In certain embodiments, the additional agent isibrutinib. In certain embodiments, the additional agent is duvelisib. Incertain embodiments, the additional agent is idelalisib. In certainembodiments, the additional agent is venetoclax. In certain embodiments,the additional agent is temozolomide.

The invention also provides, in certain embodiments, isolated nucleicacids encoding an antibodies, or antigen binding portions thereof, likethat described herein. Further, the invention includes a vectorcomprising the nucleic acid, and a host cell, e.g., a prokaryotic or aeukaryotic cell (e.g., animal cell, a protest cell, a plant cell, and afungal cell) comprising the vector. In embodiment of the invention, theanimal cell is selected from the group consisting of a mammalian cell,an insect cell, and an avian cell. In one embodiment, the mammalian cellis selected from the group consisting of a CHO cell, a COS cell, and anSp2/0 cell.

In certain embodiments, the invention features anti-hEGFR Antibody DrugConjugates (ADC) comprising an anti-hEGFR antibody conjugated to anauristatin, wherein the antibody comprises a heavy chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a heavychain CDR1 domain comprising the amino acid sequence of SEQ ID NO: 10;and a light chain CDR3 domain comprising the amino acid sequence of SEQID NO: 8, a light chain CDR2 domain comprising the amino acid sequenceof SEQ ID NO: 7, and a light chain CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In one embodiment, the antibody comprises aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 9, and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 5. In yet another embodiment, the antibodycomprises an IgG heavy chain immunoglobulin constant domain. In stillanother embodiment, the IgG is an IgG1 or an IgG4 heavy chainimmunoglobulin constant domain.

In one embodiment, the invention includes an ADC, wherein the auristatinis monomethylaurisatin F (MMAF) or monomethyauristatin E (MMAE). In oneembodiment, the invention includes an ADC, wherein the auristatin ismonomethylaurisatin F (MMAF). In one embodiment, the invention includesan ADC, wherein the auristatin is monomethyauristatin E (MMAE).

In a further embodiment, the invention includes a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 15, and comprises a light chaincomprising the amino acid sequence of SEQ ID NO: 13.

In still another embodiment of the invention, the anti-EGFR antibody iscovalently linked to the auristatin by a linker comprisingmaleimidocaproyl, valine-citrulline, p-aminobenzylalcohol (mc-vc-PABA).

In one embodiment, the invention includes an ADC comprising an anti-EGFRand a radiolabel, e.g. indium.

In one embodiment, an anti-EGFR antibody described herein is covalentlylinked to at least one pyrrolobenzodiazepine (PBD). In certainembodiments, the anti-EGFR antibody disclosed herein is linked to a PBDas described in FIG. 21 (i.e., SGD-1882).

In some embodiments, the invention features pharmaceutical compositionscomprising the ADC described herein, and a pharmaceutically acceptablecarrier In certain embodiments, the invention features pharmaceuticalscomposition comprising an ADC mixture comprising the ADC describedherein, wherein the average drug to antibody ratio (DAR) range in theADC mixture is 2 to 4. In certain embodiments, the average drug toantibody ratio (DAR) range in the ADC mixture is 2.4 to 3.6.

In one embodiment, the invention features pharmaceutical compositionscomprising an ADC mixture comprising anti-hEGFR Antibody Drug Conjugates(ADCs), and a pharmaceutically acceptable carrier, wherein the ADCmixture has an average Drug to Antibody Ratio (DAR) of 2 to 4, andwherein said ADC comprises monomethyauristatin E (MMAE) conjugated to ananti-hEGFR antibody comprising a heavy chain CDR3 domain comprising theamino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 11, and a heavy chainCDR1 domain comprising the amino acid sequence of SEQ ID NO: 10; and alight chain CDR3 domain comprising the amino acid sequence of SEQ ID NO:8, a light chain CDR2 domain comprising the amino acid sequence of SEQID NO: 7, and a light chain CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6 In one embodiment, the heavy chain variableregion of the antibody comprises the amino acid sequence set forth inSEQ ID NO: 9, and the light chain variable region of the anti-EGFRantibody comprises the amino acid sequence set forth in SEQ ID NO: 5.

In other embodiments of the invention, the antibody comprises an IgGheavy chain immunoglobulin constant domain. In further embodiments, theinvention includes an antibody having an IgG1 or an IgG4 heavy chainimmunoglobulin constant domain. In one embodiment, the inventionincludes an antibody is an IgG1 isotype.

In yet another embodiment, the invention includes antibodies comprisinga heavy chain comprising the amino acid sequence set forth in SEQ ID NO:15, and a light chain comprising the amino acid sequence of SEQ ID NO:13. In one embodiment, the invention features having an MMAE which isconjugated to the antibody by a maleimidocaproyl, val-cit, PABA linker.

In one embodiment of the invention, the invention provides methods fortreating a subject having cancer, comprising administering apharmaceutical composition comprising an antibody or ADC describedherein to the subject, such that the subject having cancer is treated.In one embodiment, the cancer is selected from the group consisting ofbreast cancer, lung cancer, a glioblastoma, prostate cancer, pancreaticcancer, colon cancer, head and neck cancer, and kidney cancer. In oneembodiment, the cancer is selected from the group consisting of breastcancer, lung cancer, a glioblastoma, prostate cancer, pancreatic cancer,colon cancer, colorectal cancer, head and neck cancer, mesothelioma,kidney cancer, squamous cell carcinoma, triple negative breast cancer,and non-small cell lung cancer. In yet another embodiment, the cancercontains amplifications of EGFR or overexpresses EGFR. In oneembodiment, the squamous cell carcinoma is squamous lung cancer orsquamous head and neck cancer. In one embodiment, the cancer is an EGFRoverexpressing cancer. In one embodiment, the cancer is characterized asEGFR amplified. In one embodiment, the cancer is breast cancer. In oneembodiment, the cancer is lung cancer. In one embodiment, the cancer isprostate cancer. In one embodiment, the cancer is pancreatic cancer. Inone embodiment, the cancer is colon cancer. In one embodiment, thecancer is head and neck cancer. In one embodiment, the cancer is kidneycancer. In one embodiment, the cancer is colorectal cancer. In oneembodiment, the cancer is mesothelioma. In one embodiment, the cancer issquamous cell carcinoma. In one embodiment, the cancer is triplenegative breast cancer. In one embodiment, the cancer is non-small celllung cancer. In certain embodiments, the squamous cell carcinoma issquamous lung cancer or squamous head and neck cancer.

In addition, in certain embodiments, the invention provides methods forinhibiting or decreasing solid tumor growth in a subject having a solidtumor, said method comprising administering the pharmaceuticalcomposition described herein to the subject having the solid tumor, suchthat the solid tumor growth is inhibited or decreased. In oneembodiment, the solid tumor is a non-small cell lung carcinoma or aglioblastoma. In yet another embodiment, the solid tumor is an EGFRvIIIpositive tumor or an EGFR-expressing solid tumor. In yet anotherembodiment, the solid tumor is an EGFR overexpressing solid tumor. Inyet another embodiment, the solid tumor is an EGFR amplified tumor. Inone embodiment, the solid tumor is a non-small cell lung carcinomahaving amplified EGFR. In one embodiment, the solid tumor is a non-smallcell lung carcinoma having EGFR overexpression. In one embodiment, thesolid tumor is a glioblastoma having amplified EGFR. In one embodiment,the solid tumor is a glioblastoma having EGFR overexpression.

In certain embodiments, the invention provides combination therapieswhereby the pharmaceutical compositions described herein areadministered to a subject in need thereof, (e.g., a subject havingcancer or a solid tumor). The pharmaceutical compositions describedherein may be administered at the same time as, prior to, or followingadministration of an additional agent or additional therapy. In certainembodiments, the additional agent is selected from the group consistingof an anti-PD1 antibody, an anti-CTLA-4 antibody, temozolomide, a bcl-xlinhibitor, and a nicotinamide phosphoribosyltransferase (NAMPT)inhibitor. In yet other embodiments, the additional agent is achemotherapeutic agent. In certain embodiments, the additional therapyis radiation. In other embodiments, the additional agent is ibrutinib(Imbruvica®, Pharmacyclics). In other embodiments, the additional agentis duvelisib. In other embodiments, the additional agent is idelalisib(Zydelig®, Gilead Sciences, Inc.). In other embodiments, the additionalagent is venetoclax (ABT-199/GDC-0199, AbbVie, Inc.). In certainembodiments, the additional agent is an anti-PD1 antibody (e.g.,pembrolizumab (Keytruda®) or nivolumab). In certain embodiments, theadditional agent is an anti-CTLA-4 antibody (e.g., ipilimumab). Incertain embodiments, the additional agent is temozolomide.

In certain embodiments, the invention features a chimeric antigenreceptor (CAR) comprising antigen binding regions, e.g. CDRs, of theantibodies described herein or an scFv described herein. In certainembodiments, the invention features a CAR comprising a variable heavylight chain comprising a CDR3 domain comprising the amino acid sequenceset forth in SEQ ID NO: 40, a CDR2 domain comprising the amino acidsequence set forth in SEQ ID NO: 39, and a CDR1 domain comprising theamino acid sequence set forth in SEQ ID NO: 38; and a heavy chainvariable region comprising a CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 37, a CDR2 domain comprising the aminoacid sequence set forth in SEQ ID NO: 36, and a CDR1 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 35.

In certain embodiments, the invention features a CAR comprising avariable heavy light chain comprising a CDR3 domain comprising the aminoacid sequence set forth in SEQ ID NO: 12, a CDR2 domain comprising theamino acid sequence set forth in SEQ ID NO: 11, and a CDR1 domaincomprising the amino acid sequence set forth in SEQ ID NO: 10; and aheavy chain variable region comprising a CDR3 domain comprising theamino acid sequence set forth in SEQ ID NO: 8, a CDR2 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 7, and a CDR1 domaincomprising the amino acid sequence set forth in SEQ ID NO: 6.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the variable heavy (VH) and variable light (VL) chainregion amino acid sequences of Ab1 (SEQ ID NOs: 1 and 5) and AbA (SEQ IDNOs: 9 and 5). CDR sequences within the VH and VL regions are boxed, anddifferences between the Ab1 VH sequence and the AbA VH sequence areshaded.

FIG. 2 describes the full length light and heavy chains for Ab1 (SEQ IDNOs: 13 and 14) and AbA (SEQ ID NOs: 13 and 15). Differences between theAb1 sequence and the AbA sequence in the heavy chain are highlighted.

FIG. 3 provides a table summarizing the Biacore binding assay affinitymeasurements for multiple Ab1 variant antibodies in comparison to Ab1and Ab2. EGFR (1-525) and EGFRvIII were used in the binding analysis.k_(a) (M⁻¹ s⁻¹) as described in FIG. 3 refers to the rate constant forassociation of an antibody to the antigen to form the antibody/antigencomplex, k_(d) (s⁻¹) refers to the off rate constant for dissociation ofan antibody from the antibody/antigen complex, and the K_(d) (M) refersto the equilibrium dissociation constant rate.

FIG. 4 provides a graphic summary of a FACS analysis showing that AbAhad improved binding to A431 tumor cells (human squamous carceinomacells) over Ab1, but a lower binding affinity in comparison to Ab2.

FIG. 5 depicts the results from a FACS competition assay that indicatesthat the Ab1 variant antibodies recognize the same EGFR epitope as Ab1.

FIG. 6 provides a summary of the binding of Ab1 and the Ab1 variantantibodies to EGFR (1-525). Closed circles represent Ab1 or Ab2(controls) and open circles represent the Ab1 variant antibodies.Circles indicate Group 1 and Group 2, summarizing data provided in FIG.7

FIGS. 7A and 7B provide results from Western blot analysis examining theactivity of Ab1 and the Ab1 variant antibodies on different cell linesin vitro. Cells from SCC-15 (FIG. 7A) and H292 cells were exposed toconditions as described in Example 4, and analyzed using Western BlotAnalysis using anti-phosphotyrosine EGFR (pY EGFR), anti-EGFR (tot EGFR(total EGFR)), and anti-actin (actin) antibodies. FIG. 7A providesresults showing the ability of Ab1, Ab2, and the Ab1 variant antibodiesto inhibit EGF-mediated tyrosine phosphorylation of EGFR in SCC-15cells. FIG. 7B provides results showing the ability of Ab1, Ab2, and theAb1 variant antibodies to inhibit EGF-mediated tyrosine phosphorylationof EGFR in H292 cells.

FIGS. 8A and 8B graphically depict results of pEGFR ELISA assaysincluding Ab1, Ab2 and the Ab1 variants (FIG. 8A) and the level ofinhibition of Ab1 in comparison to Ab2 and AbP (FIG. 8B) from the A431inhibition study. The Y-axis of FIG. 8A is the optical density (OD) at450 nm.

FIG. 9 graphically depicts binding of Ab1, Ab2, and the Ab1 variantantibodies to normal human epidermal keratinocytes expressing wild typeEGFR using FACS binding assay.

FIG. 10 graphically depicts the results of a mouse xenograft inhibitionassay comparing the ability of AbA, AbG, AbK, AbM, and AbP to inhibittumor growth in a human NSCLC carcinoma xenograft in comparison to Ab1,Ab2, and a human IgG (huIgG) control. Arrows indicate time points ofadministration of the various antibodies.

FIG. 11 provides the structure of the AbA-malemidocaproyl-vc-PABA-MMAEADC (referred to herein as “AbA-vcMMAE”).

FIGS. 12-1 and 12-2 provide results from hydrophobic interactionchromatography (HIC) analysis of the purification of AbA-vcMMAE.

FIGS. 13-1 and 13-2 provide results from size exclusion chromatography(SEC) analysis of AbA-vcMMAE.

FIGS. 14A and 14B graphically depict results from two mouse xenograftinhibition assays using anti-EGFR ADCs. FIG. 14A depicts results from amouse xenograft inhibition assay comparing the inhibition of tumorgrowth in NCI-H1703 cells from a human NSCLC carcinoma xenograftdemonstrating enhanced inhibition of AbA-vcMMAE compared to Ab1 and anAb1-mcMMAF ADC.

FIG. 14B depicts results from a mouse xenograft inhibition assaycomparing the inhibition of tumor growth in EBC-1 cells from a humanNSCLC carcinoma xenograft demonstrating an enhanced inhibition ofAbA-vcMMAE compared to Ab1 and Ab1-mcMMAF ADC. Arrows indicate timepoints of administration of the antibody.

FIGS. 15A and 15B graphically depict results from mouse xenograftinhibition assays using anti-EGFR ADCs. FIG. 15A shows results of amouse xenograft inhibition assay comparing the inhibition of tumorgrowth in NCI-H292 cells demonstrating an enhanced inhibition bypurified AbA-vcMMAE (AbA-vcMMAEp) and AbA-vcMMAE compared to Ab1-vcMMAEpurified (Ab1-vcMMAEp), Ab1-vcMMAE, Ab1-mcMMAF ADC purified(Ab1-mcMMAFp), and Ab1-mcMMAF (versus three controls). FIG. 15B showsresults of a mouse xenograft inhibition assay comparing the inhibitionof tumor growth in NCI-H292 cells demonstrating enhanced inhibitionactivity of AbA-vcMMAE compared to purified AbA-vcMMAE (AbA-vcMMAEp) andAbA-vcMMAE compared to purified Ab1-vcMMAE (Ab1-vcMMAEp), Ab1-vcMMAE,Ab1-mcMMAF, and Ab1-mcMMAFp. Doses of the molecules in FIGS. 15A and Bare indicated in parentheses, i.e., 3 mg/kg or 6 mg/kg. Arrows indicatetime points of administration of the antibody or ADC. Control 2 in FIG.15 represents a negative control which is an anti-tetanus toxin antibodywhich does not bind to EGFR.

FIGS. 16A and 16B provide the amino acid sequences of the Ab1 variantvariable heavy (VH) library design (FIG. 16A) and the Ab1 variantvariable light (VL) library design (FIG. 16B).

FIG. 17 shows a schematic of EGFR and the regions bound by Ab1 and Ab2.

FIG. 18 graphically depicts results from mouse xenograft inhibitionassays (using NCI-H292 (NSCLC) cells) using anti-EGFR ADCs. Doses of themolecules are indicated in parentheses, i.e., 3 mg/kg or 6 mg/kg. Arrowsindicate time points of administration of the antibody or ADC.

FIG. 19 graphically depicts results from a mouse glioblastoma xenograftinhibition assay using anti-EGFR MMAE and MMAF ADCs. Doses of themolecules in FIG. 19 are indicated in parentheses, i.e., 1 mg/kg. Arrowsindicate time points of administration of the antibody or ADC. Control 2in FIG. 19 represents a negative control which is an anti-tetanus toxinantibody which does not bind to EGFR.

FIGS. 20A and 20B graphically depict results from a single-photonemission computed tomography (SPECT) imaging assay comparing theefficacy of antibody uptake by EGFR expressing tumors in two tumormodels (SW48 (FIG. 20A) and EBC1 (FIG. 20B) tumor models, respectively)using AbA, Ab1, or a control antibody labelled with ¹¹¹In.

FIG. 21 depicts the structure of a PBD dimer (SGD-1882) conjugated to anantibody (Ab) via a maleimidocaproyl-valine-alanine linker (collectivelyreferred to as SGD-1910).

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the invention relate to anti-EGFR antibodies andantibody fragments, anti-EGFR ADCs, and pharmaceutical compositionsthereof, as well as nucleic acids, recombinant expression vectors andhost cells for making such antibodies and fragments. Methods of usingthe antibodies and ADCs described herein to detect human EGFR, toinhibit human EGFR activity (in vitro or in vivo), and to treat cancerssuch as epithelial cancers, breast cancer, colorectal cancer, head andneck cancers (e.g. glioblastomas), lung cancer, kidney cancer,pancreatic cancer, mesothelioma, squamous cell carcinoma (e.g., squamouslung cancer or squamous head and neck cancer), triple negative breastcancer, non-small cell lung cancer, and prostate cancer are alsoencompassed by the invention.

I. Definitions

In order that the invention may be more readily understood, certainterms are first defined. In addition, it should be noted that whenever avalue or range of values of a parameter are recited, it is intended thatvalues and ranges intermediate to the recited values are also intendedto be part of this invention.

The terms “anti-Epidermal Growth Factor (EGF) Receptor antibody” or“anti-EGFR antibody”, used interchangeably herein, refer to an antibodythat specifically binds to EGFR. An antibody “which binds” an antigen ofinterest, i.e., EGFR, is one capable of binding that antigen withsufficient affinity such that the antibody is useful in targeting a cellexpressing the antigen. In a preferred embodiment, the antibodyspecifically binds to human EGFR (hEGFR). Examples of anti-EGFRantibodies are disclosed in Example 1 below. Unless otherwise indicated,the term “anti-EGFR antibody” is meant to refer to an antibody whichbinds to wild type EGFR or any variant of EGFR, such as EGFRvIII.

The amino acid sequence of wild type human EGFR is provided below as SEQID NO: 32, where the signal peptide (amino acid residues 1-24) areunderlined, and the amino acid residues of the extracellular domain(ECD, amino acid residues 25-645) are highlighted in bold. A truncatedwild type ECD of the EGFR (also referred to herein as EGFR(1-525))corresponds to SEQ ID NO: 47 and is equivalent to amino acids 1-525 ofSEQ ID NO: 32. The mature form of wild type EGFR corresponds to theprotein without the signal peptide, i.e., amino acid residues 25 to 1210of SEQ ID NO: 32.

(SEQ ID NO: 32) 1 mrpsgtagaa llallaalcp asra leekkv cqgtsnkltqlgtfedhfls lqrmfnncev 61 vlgnleityv qrnydlsflk tiqevagyvl ialntveriplenlqiirgn myyensyala 121 vlsnydankt glkelpmrnl qeilhgavrf snnpalcnvesiqwrdivss dflsnmsmdf 181 qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcsgrcrgkspsd cchnqcaagc 241 tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvnpegkysfgat cvkkcprnyv 301 vtdhgscvra cgadsyemee dgvrkckkce gpcrkvcngigigefkdsls inatnikhfk 361 nctsisgdlh ilpvafrgds fthtppldpq eldilktvkeitgflliqaw penrtdlhaf 421 enleiirgrt kqhgqfslav vslnitslgl rslkeisdgdviisgnknlc yantinwkkl 481 fgtsgqktki isnrgensck atgqvchalc spegcwgpeprdcvscrnvs rgrecvdkcn 541 llegeprefv enseciqchp eclpqamnit ctgrgpdnciqcahyidgph cvktcpagvm 601 genntlvwky adaghvchlc hpnctygctg pglegcptngpkipsiatgm vgalllllvv 661 algiglfmrr rhivrkrtlr rllqerelve pltpsgeapnqallrilket efkkikvlgs 721 gafgtvykgl wipegekvki pvaikelrea tspkankeildeayvmasvd nphvcrllgi 781 cltstvqlit qlmpfgclld yvrehkdnig sqyllnwcvqiakgmnyled rrlvhrdlaa 841 rnvlvktpqh vkitdfglak llgaeekeyh aeggkvpikwmalesilhri ythqsdvwsy 901 gvtvwelmtf gskpydgipa seissilekg erlpqppictidvymimvkc wmidadsrpk 961 freliiefsk mardpqrylv iqgdermhlp sptdsnfyralmdeedmddv vdadeylipq 1021 qgffsspsts rtpllsslsa tsnnstvaci drnglqscpikedsflqrys sdptgalted 1081 siddtflpvp eyinqsvpkr pagsvqnpvy hnqplnpapsrdphyqdphs tavgnpeyln 1141 tvqptcvnst fdspahwaqk gshqisldnp dyqqdffpkeakpngifkgs taenaeylrv 1201 apqssefigaThe amino acid sequence of the ECD of human EGFR is provided below asSEQ ID NO: 34, and includes the signal sequence (underlined).

(SEQ ID NO: 34) 1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltqlgtfedhfls lqrmfnncev 61 vlgnleityv qrnydlsflk tiqevagyvl ialntveriplenlqiirgn myyensyala 121 vlsnydankt glkelpmrnl qeilhgavrf snnpalcnvesiqwrdivss dflsnmsmdf 181 qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcsgrcrgkspsd cchnqcaagc 241 tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvnpegkysfgat cvkkcprnyv 301 vtdhgscvra cgadsyemee dgvrkckkce gpcrkvcngigigefkdsls inatnikhfk 361 nctsisgdlh ilpvafrgds fthtppldpq eldilktvkeitgflliqaw penrtdlhaf 421 enleiirgrt kqhgqfslav vslnitslgl rslkeisdgdviisgnknlc yantinwkkl 481 fgtsgqktki isnrgensck atgqvchalc spegcwgpeprdcvscrnvs rgrecvdkcn 541 llegeprefv enseciqchp eclpqamnit ctgrgpdnciqcahyidgph cvktcpagvm 601 genntlvwky adaghvchlc hpnctygctg pglegcptngpkips The overall structure of EGFR is described in FIG. 17. The ECD of EGFRhas four domains (Cochran et al. (2004) J. Immunol. Methods, 287,147-158). Domains I and III have been suggested to contribute to theformation of high affinity binding sites for ligands. Domains II and IVare cysteine rich, laminin-like regions that stabilize protein foldingand contain a possible EGFR dimerization interface.

EGFR variants may result from gene rearrangement accompanied by EGFRgene amplification. EGFRvIII is the most commonly occurring variant ofthe EGFR in human cancers (Kuan et al. Endocr Relat Cancer. 8(2):83-96(2001)). During the process of gene amplification, a 267 amino aciddeletion occurs in the extracellular domain of EGFR with a glycineresidue inserted at the fusion junction. Thus, EGFRvIII lacks aminoacids 6-273 of the extracellular domain of wild type EGFR and includes aglycine residue insertion at the junction. The EGFRvIII variant of EGFRcontains a deletion of 267 amino acid residues in the extracellulardomain where a glycine is inserted at the deletion junction. TheEGFRvIII amino acid sequence is shown below as SEQ ID NO: 33 (the ECD ishighlighted in bold and corresponds to SEQ ID NO: 46 the signal sequenceis underlined).

(SEQ ID NO: 33) mrpsgtagaallallaalcpasra leekkgnyvvtdhgscvracgadsyemeedgvrkckkcegperkvcngigigefkdslsinatnikhfknctsisgdlhilpvafrgdsfthtppldpqeldilktvkeitgflliqawpenrtdlhafenleiirgrtkqhgqfslavvslnitslglrslkeisdgdviisgnknlcyantinwkklfgtsgqktkiisnrgensckatgqvchalcspegcwgpeprdcvscrnvsrgrecvdkcnllegeprefvenseciqchpeclpqamnitctgrgpdnciqcahyidgphcvktcpagvmgenntlvwkyadaghvchlchpnctygctgpglegcptngpkipsiatgmvgalllllvvalgiglfmrrrhivrkrtlrrllqerelvepltpsgeapnqallrilketefkkikvlgsgafgtvykglwipegekvkipvaikelreatspkankeildeayvmasvdnphvcrllgicltstvqlitqlmpfgclldyvrehkdnigsqyllnwcvqiakgmnyledrrlvhrdlaarnvlvktpqhvkitdfglakllgaeekeyhaeggkvpikwmalesilhriythqsdvwsygvtvwelmtfgskpydgipaseissilekgerlpqppictidvymimvkcwmidadsrpkfreliiefskmardpqrylviqgdermhlpsptdsnfyralmdeedmddvvdadeylipqqgffsspstsrtpllsslsatsnnstvacidrnglqscpikedsflqryssdptgaltedsiddtflpvpeyinqsvpkrpagsvgnpvyhnqplnpapsrdphyqdphstavgnpeylntvqptcvnstfdspahwaqkgshqisldnpdyqqdffpkeakpngifkgstaenaeylrvapqssefiga

EGFRvIII contributes to tumor progression through constitutive signalingin a ligand independent manner. EGFRvIII is not known to be expressed innormal tissues (Wikstrand et al. Cancer Research 55(14): 3140-3148(1995); Olapade-Olaopa et al. Br J Cancer. 82(1):186-94 (2000)), butshows significant expression in tumor cells, including breast cancers,gliomas, NSCL cancers, ovarian cancers, and prostate cancers (Wikstrandet al. Cancer Research 55(14): 3140-3148 (1995); Ge et al. Int J Cancer.98(3):357-61 (2002); Wikstrand et al. Cancer Research 55(14): 3140-3148(1995); Moscatello et al. Cancer Res. 55(23):5536-9 (1995); Garcia dePalazzo et al. Cancer Res. 53(14):3217-20 (1993); Moscatello et al.Cancer Res. 55(23):5536-9 (1995); and Olapade-Olaopa et al. 2(1):186-94(2000)).

“Biological activity of EGFR” as used herein, refers to all inherentbiological properties of the EGFR, including, but not limited to,binding to epidermal growth factor (EGF), binding to tumor growth factorα (TGFα), homodimerization, activation of JAK2 kinase activity,activation of MAPK kinase activity, and activation of transmembranereceptor protein tyrosine kinase activity.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody or an ADC with a secondchemical species, mean that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on the chemical species; for example, an antibody recognizesand binds to a specific protein structure rather than to proteinsgenerally. If an antibody or ADC is specific for epitope “A”, thepresence of a molecule containing epitope A (or free, unlabeled A), in areaction containing labeled “A” and the antibody, will reduce the amountof labeled A bound to the antibody or ADC.

The phrase “specifically binds to hEGFR” or “specific binding to hEGFR”,as used herein, refers to the ability of an anti-EGFR antibody or ADC tointeract with hEGFR with an affinity equal to or greater than that ofAb1 or an Ab1 ADC.

The term “specific binding to EGFR(1-525)” or “specifically binds toEGFR(1-525),” as used herein, refers to an antibody or an ADC that bindsto EGFR(1-525) and has a dissociation constant (K_(D)) of 2.3×10⁻⁶ M orless, as determined by surface plasmon resonance.

The term “antibody” broadly refers to an immunoglobulin (Ig) molecule,generally comprised of four polypeptide chains, two heavy (H) chains andtwo light (L) chains, or any functional fragment, mutant, variant, orderivative thereof, that retains the essential target binding featuresof an Ig molecule. Such mutant, variant, or derivative antibody formatsare known in the art. Non-limiting embodiments of which are discussedbelow.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY) and class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1and IgA2) or subclass.

The term “antigen binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hIL-13). It has been shown that the antigen binding function ofan antibody can be performed by fragments of a full-length antibody.Such antibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antigen binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publicationWO 90/05144 A1 herein incorporated by reference), which comprises asingle variable domain; and (vi) an isolated complementarity determiningregion (CDR). Furthermore, although the two domains of the Fv fragment,VL and VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.Acad. Sci. USA 85:5879-5883). Such single chain antibodies are alsointended to be encompassed within the term “antigen binding portion” ofan antibody. In certain embodiments of the invention, scFv molecules maybe incorporated into a fusion protein. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123). Such antibody binding portions are knownin the art (Kontermann and Dubel eds., Antibody Engineering (2001)Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

The term “antibody construct” as used herein refers to a polypeptidecomprising one or more the antigen binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holliger,P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constantdomain refers to a heavy or light chain constant domain. Exemplary humanIgG heavy chain and light chain constant domain amino acid sequences areknown in the art and represented below.

Sequence of Human IgG Heavy Chain Constant Domain and Light ChainConstant Domain

Sequence Sequence Protein Identifier 12345678901234567890123456789012Ig gamma-1 SEQ ID NO: 41 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYconstant region FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig gamma-1 SEQ ID NO: 42 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYconstant region FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS mutantLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig Kappa constant SEQ ID NO: 43RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF region YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Ig Lambda SEQ ID NO:44QPKAAPSVTLFPPSSEELQANKATLVCLISDF constant regionYPGAVTVAWKADSSPVKAGVETTTPSKQSNNK YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

Still further, an antibody or antigen binding portion thereof may bepart of a larger immunoadhesion molecules, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionmolecules can be obtained using standard recombinant DNA techniques, asdescribed herein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds EGFR is substantially free of antibodies that specifically bindantigens other than EGFR). An isolated antibody that specifically bindsEGFR may, however, have cross-reactivity to other antigens, such as EGFRmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a nonhuman species (e.g.,a mouse) but in which at least a portion of the VH and/or VL sequencehas been altered to be more “human-like”, i.e., more similar to humangermline variable sequences. In particular, the term “humanizedantibody” is an antibody or a variant, derivative, analog or fragmentthereof which immunospecifically binds to an antigen of interest andwhich comprises a framework (FR) region having substantially the aminoacid sequence of a human antibody and a complementary determining region(CDR) having substantially the amino acid sequence of a non-humanantibody. As used herein, the term “substantially” in the context of aCDR refers to a CDR having an amino acid sequence at least 80%,preferably at least 85%, at least 90%, at least 95%, at least 98% or atleast 99% identical to the amino acid sequence of a non-human antibodyCDR. A humanized antibody comprises substantially all of at least one,and typically two, variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) inwhich all or substantially all of the CDR regions correspond to those ofa non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. In some embodiments, ahumanized antibody contains both the light chain as well as at least thevariable domain of a heavy chain. The antibody also may include the CH1,hinge, CH2, CH3, and CH4 regions of the heavy chain. In someembodiments, a humanized antibody only contains a humanized light chain.In other embodiments, a humanized antibody only contains a humanizedheavy chain. In specific embodiments, a humanized antibody only containsa humanized variable domain of a light chain and/or humanized heavychain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The terms “Kabat numbering,” “Kabat definitions,” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain (HC) and the light chain(LC), which are designated CDR1, CDR2 and CDR3 (or specifically HC CDR1,HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3), for each of thevariable regions. The term “CDR set” as used herein refers to a group ofthree CDRs that occur in a single variable region capable of binding theantigen. The exact boundaries of these CDRs have been defineddifferently according to different systems. The system described byKabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) notonly provides an unambiguous residue numbering system applicable to anyvariable region of an antibody, but also provides precise residueboundaries defining the three CDRs. These CDRs may be referred to asKabat CDRs. Chothia and coworkers (Chothia &Lesk, J. Mol. Biol.196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) foundthat certain sub-portions within Kabat CDRs adopt nearly identicalpeptide backbone conformations, despite having great diversity at thelevel of amino acid sequence. These sub-portions were designated as L1,L2 and L3 or H1, H2 and H3 where the “L” and the “H” designates thelight chain and the heavy chains regions, respectively. These regionsmay be referred to as Chothia CDRs, which have boundaries that overlapwith Kabat CDRs. Other boundaries defining CDRs overlapping with theKabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) andMacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughpreferred embodiments use Kabat or Chothia defined CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In a preferred embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. In one embodiment, the invention includes anamino acid sequence having at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1to 31, 35-40, or 50 to 85.

The term “multivalent antibody” is used herein to denote an antibodycomprising two or more antigen binding sites. In certain embodiments,the multivalent antibody may be engineered to have the three or moreantigen binding sites, and is generally not a naturally occurringantibody.

The term “multispecific antibody” refers to an antibody capable ofbinding two or more unrelated antigens. In one embodiment, themultispecific antibody is a bispecific antibody that is capable ofbinding to two unrelated antigens, e.g., a bispecific antibody, orantigen-binding portion thereof, that binds EGFR (e.g., EGFRvIII) andCD3.

The term “dual variable domain” or “DVD,” as used interchangeablyherein, are antigen binding proteins that comprise two or more antigenbinding sites and are tetravalent or multivalent binding proteins. SuchDVDs may be monospecific, i.e., capable of binding one antigen ormultispecific, i.e. capable of binding two or more antigens. DVD bindingproteins comprising two heavy chain DVD polypeptides and two light chainDVD polypeptides are referred to a DVD Ig. Each half of a DVD Igcomprises a heavy chain DVD polypeptide, and a light chain DVDpolypeptide, and two antigen binding sites. Each binding site comprisesa heavy chain variable domain and a light chain variable domain with atotal of 6 CDRs involved in antigen binding per antigen binding site. Inone embodiment, the CDRs described herein are used in an anti-EGFR DVD.

The term “chimeric antigen receptor” or “CAR” refers to a recombinantprotein comprising at least (1) an antigen-binding region, e.g., avariable heavy or light chain of an antibody, (2) a transmembrane domainto anchor the CAR into a T cell, and (3) one or more intracellularsignaling domains.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody or ADC for an antigen, for example,an anti-hEGFR antibody that binds to an hEGFR antigen and/or theneutralizing potency of an antibody, for example, an anti-hEGFR antibodywhose binding to hEGFR inhibits the biological activity of hEGFR, e.g.,inhibition of phosphorylation of EGFR in an EGFR expressing cell line,e.g., the human lung carcinoma cell line H292, or inhibition ofproliferation of EGFR expressing cell lines, e.g., human H292 lungcarcinoma cells, human H1703 lung carcinoma cells, or human EBC1 lungcarcinoma cells.

The term “non small-cell lung carcinoma (NSCLC) xenograft assay,” asused herein, refers to an in vivo assay used to determine whether ananti-EGFR antibody or ADC, can inhibit tumor growth (e.g., furthergrowth) and/or decrease tumor growth resulting from the transplantationof NSCLC cells into an immunodeficient mouse. An NSCLC xenograft assayincludes transplantation of NSCLC cells into an immunodeficient mousesuch that a tumor grows to a desired size, e.g., 200-250 mm³, whereuponthe antibody or ADC is administered to the mouse to determine whetherthe antibody or ADC can inhibit and/or decrease tumor growth. In certainembodiments, the activity of the antibody or ADC is determined accordingto the percent tumor growth inhibition (% TGI) relative to a controlantibody, e.g., a human IgG antibody (or collection thereof) which doesnot specifically bind tumor cells, e.g., is directed to an antigen notassociated with cancer or is obtained from a source which isnoncancerous (e.g., normal human serum). In such embodiments, theantibody (or ADC) and the control antibody are administered to the mouseat the same dose, with the same frequency, and via the same route. Inone embodiment, the mouse used in the NSCLC xenograft assay is a severecombined immunodeficiency (SCID) mouse and/or an athymic CD-1 nudemouse. Examples of NSCLC cells that may be used in the NSCLC xenograftassay include, but are not limited to, H292 cells (e.g., NCIH292 [H292](ATCC® CRL1848™).

The term “epitope” refers to a region of an antigen that is bound by anantibody or ADC. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments,may have specific three dimensional structural characteristics, and/orspecific charge characteristics. In certain embodiments, an antibody issaid to specifically bind an antigen when it preferentially recognizesits target antigen in a complex mixture of proteins and/ormacromolecules. In a particular embodiment, the antibodies of theinvention bind to an epitope defined by the amino acid sequenceCGADSYEMEEDGVRKC (SEQ ID NO: 45) (which corresponds to amino acidresidues 287-302 of the mature form of hEGFR).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Jöhnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277. In one embodiment, surface plasmonresonance is determined according to the methods described in Example 2

The term “k_(on)” or “k_(a)”, as used herein, is intended to refer tothe on rate constant for association of an antibody to the antigen toform the antibody/antigen complex.

The term “k_(off)” or “k_(d)”, as used herein, is intended to refer tothe off rate constant for dissociation of an antibody from theantibody/antigen complex.

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction (e.g., AbA antibody and EGFR). K_(D) is calculated byk_(a)/k_(d).

The term “competitive binding”, as used herein, refers to a situation inwhich a first antibody competes with a second antibody, for a bindingsite on a third molecule, e.g., an antigen. In one embodiment,competitive binding between two antibodies is determined using FACSanalysis.

The term “competitive binding assay” is an assay used to determinewhether two or more antibodies bind to the same epitope. In oneembodiment, a competitive binding assay is a competition fluorescentactivated cell sorting (FACS) assay which is used to determine whethertwo or more antibodies bind to the same epitope by determining whetherthe fluorescent signal of a labeled antibody is reduced due to theintroduction of a non-labeled antibody, where competition for the sameepitope will lower the level of fluorescence. An example of acompetition binding FACS assay is provided in Example 3 wherecompetition FACS assay is described using U87MG cells (which expressEGFRvIII).

The term “labeled antibody” as used herein, refers to an antibody, or anantigen binding portion thereof, with a label incorporated that providesfor the identification of the binding protein, e.g., an antibody.Preferably, the label is a detectable marker, e.g., incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (e.g., streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). Examples of labels forpolypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho or ¹⁵³Sm); fluorescent labels (e.g., FITC,rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradishperoxidase, luciferase, alkaline phosphatase); chemiluminescent markers;biotinyl groups; predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags); andmagnetic agents, such as gadolinium chelates.

The term “antibody-drug-conjugate” or “ADC” refers to a binding protein,such as an antibody or antigen binding fragment thereof, chemicallylinked to one or more chemical drug(s) (also referred to herein asagent(s)) that may optionally be therapeutic or cytotoxic agents. In apreferred embodiment, an ADC includes an antibody, a cytotoxic ortherapeutic drug, and a linker that enables attachment or conjugation ofthe drug to the antibody. An ADC typically has anywhere from 1 to 8drugs conjugated to the antibody, including drug loaded species of 2, 4,6, or 8. Non-limiting examples of drugs that may be included in the ADCsare mitotic inhibitors, antitumor antibiotics, immunomodulating agents,vectors for gene therapy, alkylating agents, antiangiogenic agents,antimetabolites, boron-containing agents, chemoprotective agents,hormones, antihormone agents, corticosteroids, photoactive therapeuticagents, oligonucleotides, radionuclide agents, topoisomerase inhibitors,tyrosine kinase inhibitors, and radiosensitizers.

The terms “anti-Epidermal Growth Factor antibody drug conjugate,”“anti-EGFR antibody drug conjugate,” or “anti-EGFR ADC”, usedinterchangeably herein, refer to an ADC comprising an antibody thatspecifically binds to EGFR, whereby the antibody is conjugated to one ormore chemical agent(s). In one embodiment, the anti-EGFR ADC is antibodyAbA conjugated to an auristatin, e.g., MMAE or MMAF. Amino acidsequences corresponding to the light and heavy chains of antibody AbAare provided in SEQ ID NO: 13 and SEQ ID NO: 15, respectively.

The term “auristatin”, as used herein, refers to a family of antimitoticagents. Auristatin derivatives are also included within the definitionof the term “auristatin”. Examples of auristatins include, but are notlimited to, auristatin E (AE), monomethylauristatin E (MMAE),monomethylauristatin F (MMAF), and synthetic analogs of dolastatin. Inone embodiment, an anti-EGFR antibody described herein is conjugated toan auristatin to form an anti-EGFR ADC.

As used herein, the term “AbA-vcMMAE” is used to refer to an ADCcomprising the antibody AbA coupled to monomethylauristatin E (MMAE) viaa maleimidocaproyl valine citrulline p-aminobenzyloxycarbamyl (PABA)linker. AbA-vcMMAE is described in FIG. 11.

As used herein, the term “mcMMAF” is used to refer to a linker/drugcombination of maleimidocaproyl-monomethylauristatin F (MMAF).

The term “drug-to-antibody ratio” or “DAR” refers to the number ofdrugs, e.g., auristatin, attached to the antibody of the ADC. The DAR ofan ADC can range from 1 to 8, although higher loads, e.g., 10, are alsopossible depending on the number of linkage site on an antibody. Theterm DAR may be used in reference to the number of drugs loaded onto anindividual antibody, or, alternatively, may be used in reference to theaverage or mean DAR of a group of ADCs.

The term “undesired ADC species”, as used herein, refers to any drugloaded species which is to be separated from an ADC species having adifferent drug load. In one embodiment, the term undesired ADC speciesmay refer to drug loaded species of 6 or more, i.e., ADCs with a DAR of6 or more, including DARE, DART, DAR8, and DAR greater than 8 (i.e.,drug loaded species of 6, 7, 8, or greater than 8). In a separateembodiment, the term undesired ADC species may refer to drug loadedspecies of 8 or more, i.e., ADCs with a DAR of 8 or more, includingDAR8, and DAR greater than 8 (i.e., drug loaded species of 8, or greaterthan 8).

The term “ADC mixture”, as used herein, refers to a compositioncontaining a heterogeneous DAR distribution of ADCs. In one embodiment,an ADC mixture contains ADCs having a distribution of DARs of 1 to 8,e.g., 2, 4, 6, and 8 (i.e., drug loaded species of 2, 4, 6, and 8).Notably, degradation products may result such that DARs of 1, 3, 5, and7 may also be included in the mixture. Further, ADCs within the mixturemay also have DARs greater than 8. The ADC mixture results frominterchain disulfide reduction followed by conjugation. In oneembodiment, the ADC mixture comprises both ADCs with a DAR of 4 or less(i.e., a drug loaded species of 4 or less) and ADCs with a DAR of 6 ormore (i.e., a drug loaded species of 6 or more).

The term “cancer” is meant to refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include glioblastoma, non-small celllung cancer, lung cancer, colon cancer, colorectal cancer, head and neckcancer, breast cancer (e.g., triple negative breast cancer), pancreaticcancer, squamous cell tumors, squamous cell carcinoma (e.g., squamouscell lung cancer or squamous cell head and neck cancer), anal cancer,skin cancer, and vulvar cancer. In one embodiment, the antibodies orADCs of the invention are administered to a patient having a tumor(s)containing amplifications of the EGFR gene, whereby the tumor expressesthe truncated version of the EGFR, EGFRvIII. In one embodiment, theantibodies or ADCs of the invention are administered to a patient havinga solid tumor which is likely to over-express EGFR. In one embodiment,the antibodies or ADCs of the invention are administered to a patienthaving squamous cell Non-Small Cell Lung Cancer (NSCLC). In oneembodiment, the antibodies or ADCs of the invention are administered toa patient having solid tumors, including advanced solid tumors.

The term “EGFR expressing tumor,” as used herein, refers to a tumorwhich expresses EGFR protein. In one embodiment, EGFR expression in atumor is determined using immunohistochemical staining of tumor cellmembranes, where any immunohistochemical staining above background levelin a tumor sample indicates that the tumor is an EGFR expressing tumor.Methods for detecting expression of EGFR in a tumor are known in theart, e.g., the EGFR pharmDx™ Kit (Dako). In contrast, an “EGFR negativetumor” is defined as a tumor having an absence of EGFR membrane stainingabove background in a tumor sample as determined by immunohistochemicaltechniques.

The term “EGFRvIII positive tumor,” as used herein, refers to a tumorwhich expresses EGFRvIII protein. In one embodiment, EGFRvIII expressionin a tumor is determined using immunohistochemical staining of tumorcell membranes, where any immunohistochemical staining above backgroundlevel in a tumor sample indicates that the tumor is an EGFRvIIIexpressing tumor. Methods for detecting expression of EGFR in a tumorare known in the art, and include immunohistochemical assays. Incontrast, an “EGFRvIII negative tumor” is defined as a tumor having anabsence of EGFRvIII membrane staining above background in a tumor sampleas determined by immunohistochemical techniques.

The terms “overexpress,” “overexpression,” or “overexpressed”interchangeably refer to a gene that is transcribed or translated at adetectably greater level, usually in a cancer cell, in comparison to anormal cell. Overexpression therefore refers to both overexpression ofprotein and RNA (due to increased transcription, post transcriptionalprocessing, translation, post translational processing, alteredstability, and altered protein degradation), as well as localoverexpression due to altered protein traffic patterns (increasednuclear localization), and augmented functional activity, e.g., as in anincreased enzyme hydrolysis of substrate. Thus, overexpression refers toeither protein or RNA levels. Overexpression can also be by 50%, 60%,70%, 80%, 90% or more in comparison to a normal cell or comparison cell.In certain embodiments, the anti-EGFR antibodies or ADCs of theinvention are used to treat solid tumors likely to overexpress EGFR.

The term “administering” as used herein is meant to refer to thedelivery of a substance (e.g., an anti-EGFR antibody or ADC) to achievea therapeutic objective (e.g., the treatment of an EGFR-associateddisorder). Modes of administration may be parenteral, enteral andtopical. Parenteral administration is usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The term “combination therapy”, as used herein, refers to theadministration of two or more therapeutic substances, e.g., an anti-EGFRantibody or ADC and an additional therapeutic agent. The additionaltherapeutic agent may be administered concomitant with, prior to, orfollowing the administration of the anti-EGFR antibody or ADC.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to the amount of a drug, e.g., an antibody orADC, which is sufficient to reduce or ameliorate the severity and/orduration of a disorder, e.g., cancer, or one or more symptoms thereof,prevent the advancement of a disorder, cause regression of a disorder,prevent the recurrence, development, onset or progression of one or moresymptoms associated with a disorder, detect a disorder, or enhance orimprove the prophylactic or therapeutic effect(s) of another therapy(e.g., prophylactic or therapeutic agent). The effective amount of anantibody or ADC may, for example, inhibit tumor growth (e.g., inhibit anincrease in tumor volume), decrease tumor growth (e.g., decrease tumorvolume), reduce the number of cancer cells, and/or relieve to someextent one or more of the symptoms associated with the cancer. Theeffective amount may, for example, improve disease free survival (DFS),improve overall survival (OS), or decrease likelihood of recurrence.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. Anti-EGFR Antibodies

One aspect of the invention provides anti-EGFR antibodies, or antigenbinding portions thereof, having improved characteristics, e.g.,increased binding affinity for EGFR, over Ab1 and other antibodies knownin the art. Another aspect of the invention features antibody drugconjugates (ADCs) comprising an anti-EGFR antibody described herein andat least one drug(s), such as, but not limited to, an auristatin. Theantibodies or ADCs of the invention have characteristics including, butnot limited to, binding to tumor cells expressing EGFRvIII, binding towild type EGFR on tumor cells expressing EGFR, recognizing the epitopeCGADSYEMEEDGVRKC (SEQ ID NO: 45) on EGFR, binding to EGFR on normalhuman epithelial keratinocytes, and decreasing or inhibiting xenografttumor growth in a mouse model.

Ab1 (Antibody 1) is a humanized anti-EGFR antibody. The light and heavychain sequences of Ab1 are described in SEQ ID NO: 13 and SEQ ID NO: 14,respectively (see also US Patent Application Publication No.20120183471, incorporated by reference herein). The light chain variableregion of Ab1 is described in SEQ ID NO: 5, and comprises a CDR1 aminoacid sequence set forth in SEQ ID NO: 6, a CDR2 amino acid sequence setforth in SEQ ID NO: 7, and a CDR3 amino acid sequence set forth in SEQID NO: 8. The heavy chain variable region of Ab1 is described in SEQ IDNO: 1, and comprises a CDR1 amino acid sequence set forth in SEQ ID NO:2, a CDR2 amino acid sequence set forth in SEQ ID NO: 3, and a CDR3amino acid sequence set forth in SEQ ID NO: 4.

Generally, the Ab1 variant antibodies of the invention retain theepitope specificity of parental antibody Ab1. Thus, in one embodiment,the anti-EGFR antibodies of the invention are capable of binding anepitope in EGFR defined by SEQ ID NO: 45 and/or are able to compete withAb1 for binding to EGFR. In various embodiments, the binding may beassayed according to the protocol set forth in Example 3 below. In apreferred embodiment of the invention, the anti-EGFR antibodies competewith Ab1 and have an improved binding affinity, e.g., dissociationconstant (K_(a)) of between about 1×10⁻⁶ M and about 1×10⁻¹⁰ M, asdetermined by surface plasmon resonance, to 1-525 of EGFR (SEQ ID NO:47).

In one embodiment, the invention features anti-EGFR antibodies which arevariants of Ab1 and have improved characteristics, e.g., improvedbinding affinity and the ability to inhibit NSCLC tumor cellproliferation in vivo, as described in the Examples below. Collectivelythese novel antibodies are referred to herein as “Ab1 variantantibodies.” Generally, the Ab1 variant antibodies retain the sameepitope specificity as Ab1. Thus, in one embodiment, anti-EGFRantibodies, or antigen binding portions thereof, of the invention bindto an epitope within the amino acid sequence set forth in SEQ ID NO: 45and compete with an anti-EGFR antibody comprising a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO: 1 anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 5 for binding to EGFRvIII in a competitive bindingassay. In contrast to Ab1, the anti-EGFR antibodies of the invention areable to inhibit or decrease tumor growth in vivo in an H292 humannon-small cell lung carcinoma (NSCLC) xenograft assay in a nude mouseand/or bind to wild type EGFR on normal human epithelial keratinocytes.In various embodiments, anti-EGFR antibodies, or antigen bindingfragments thereof, of the invention are capable of modulating abiological function of EGFR. In other embodiments of the foregoingaspects, the anti-EGFR antibody, or antigen binding fragment thereof,binds EGFRvIII, binds EGFR on cells overexpressing EGFR, and recognizesthe epitope CGADSYEMEEDGVRKC (SEQ ID NO: 45) on EGFR. In a furtherembodiment, the anti-EGFR antibody, or antigen binding fragment thereof,binds EGFRvIII at an epitope which is distinct from the EGFRvIIIjunctional peptide. In additional embodiments of the foregoing aspects,the anti-EGFR antibody, or antigen binding fragment thereof, does notcompete with cetuximab for binding of EGFR. The AbA antibody and the Ab1variants described in the examples below have the foregoingcharacteristics.

Thus, the invention includes anti-EGFR antibodies, or antigen bindingportions thereof, that can compete with Ab1 in a competitive bindingassay but are more effective at inhibiting or decreasing tumor growth.In one embodiment, anti-EGFR antibodies, or antigen binding portionsthereof, of the invention are capable of binding to an epitope withinthe amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45) and competingwith Ab1 (or an anti-EGFR antibody comprises a heavy chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO: 1 anda light chain variable domain comprising the amino acid sequence setforth in SEQ ID NO: 5) for binding to epidermal growth factor receptorvariant III (EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay.

In one embodiment, the anti-EGFR antibodies, or antigen binding portionsthereof, of the invention bind to EGFR(1-525) (SEQ ID NO: 47) with adissociation constant (K_(d)) of about 1×10⁻⁶ M or less, as determinedby surface plasmon resonance. Alternatively, the antibodies, or antigenbinding portions thereof, bind to EGFR (1-525) (SEQ ID NO: 47) with aK_(d) of between about 1×10⁻⁶ M and about 1×10⁻¹⁰ M, as determined bysurface plasmon resonance. In a further alternative, antibodies, orantigen binding portions thereof, bind to EGFR (1-525) (SEQ ID NO: 47)with a K_(d) of between about 1×10⁻⁶ M and about 1×10⁻⁷M, as determinedby surface plasmon resonance. Alternatively, antibodies, or antigenbinding portions thereof, of the invention binds to EGFR (1-525) (SEQ IDNO: 47) with a K_(d) of between about 1×10⁻⁶ M and about 5×10⁻¹⁰ M; aK_(d) of between about 1×10⁻⁶ M and about 1×10⁻⁹M; a K_(d) of betweenabout 1×10⁻⁶ M and about 5×10⁻⁹M; a K_(d) of between about 1×10⁻⁶ M andabout 1×10⁻⁸ M; a K_(d) of between about 1×10⁻⁶ M and about 5×10⁻⁸M; aK_(d) of between about 5.9×10⁻⁷ M and about 1.7×10⁻⁹M; a K_(d) ofbetween about 5.9×10⁻⁷ M and about 2.2×10⁻⁷M, as determined by surfaceplasmon resonance. In certain embodiments, the dissociation constant(K_(d)) of the antibodies and antigen-binding fragments of the inventionis, in one embodiment, lower than the dissociation constant for Ab1 buthigher than the rate of anti-EGFR antibody cetuximab.

One advantage of the anti-EGFR antibodies, and antigen-binding portionsthereof, of the invention is that the antibodies are capable of bindingto tumor cells expressing EGFRvIII. While EGFRvIII is associated withcertain types of cancer, many anti-EGFR antibodies known in the art,e.g., cetuximab, are not effective at inhibiting or decreasing tumorgrowth in EGFRvIII expressing tumors. Thus, in one embodiment, theantibodies, or antigen binding portions thereof, of the invention bindto EGFRvIII (SEQ ID NO: 33) with a K_(d) of about 8.2×10⁻⁹ M or less, asdetermined by surface plasmon resonance. Alternatively, the antibodies,or antigen binding portions thereof, of the invention bind to EGFRvIII(SEQ ID NO: 33) with a K_(d) of between about 8.2×10⁻⁹ M and about6.3×10⁻¹⁰ M; a K_(d) of between about 8.2×10⁻⁹ M and about 2.0×10⁻⁹ M; aK_(d) of between about 2.3×10⁻⁹ M and about 1.5×10⁻¹⁰ M, as determinedby surface plasmon resonance.

The antibodies of the invention are able, in one embodiment, to inhibitor decrease tumor growth in an in vivo xenograft mouse model. Forexample, the antibodies, or antigen binding portions thereof, of theinvention are able to inhibit tumor growth by at least about 50% in anin vivo human non-small-cell lung carcinoma (NSCLC) xenograft assayrelative to a human IgG antibody which is not specific for EGFR. Incertain embodiments, the antibodies, or antigen binding portionsthereof, of the invention are able to inhibit or decrease tumor growthin an in vivo human non-small-cell lung carcinoma (NSCLC) xenograftassay relative to a human IgG antibody which is not specific for EGFR byat least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, or at least about 80%, when administeredat the same dose and dosing periodicity. In certain embodiments, theantibodies, or antigen-binding portions thereof, of the invention areable to inhibit or decrease tumor growth in an in vivo humannon-small-cell lung carcinoma (NSCLC) xenograft assay relative to ahuman IgG antibody which is not specific for EGFR by from about 80% toabout 90%, or from about 84% to about 90%, or from about 88% to about90%, when administered at the same dose and dosing periodicity.

The term a “xenograft assay”, as used herein, refers to a human tumorxenograft assay, wherein human tumor cells are transplanted, eitherunder the skin or into the organ type in which the tumor originated,into immunocompromised mice that do not reject human cells.

It should be noted that anti-EGFR antibodies, or antigen bindingportions thereof, having combinations of the aforementionedcharacteristics are also considered to be embodiments of the invention.For example, antibodies of the invention may bind to EGFR(1-525) (SEQ IDNO: 47) with a dissociation constant (K_(d)) of about 1×10⁻⁶ M or less,as determined by surface plasmon resonance, and bind to an epitopewithin the amino acid sequence CGADSYEMEEDGVRKC (SEQ ID NO: 45) andcompete with Ab1 (or an anti-EGFR antibody comprising a heavy chainvariable domain comprising the amino acid sequence set forth in SEQ IDNO: 1 and a light chain variable domain comprising the amino acidsequence set forth in SEQ ID NO: 5) for binding to epidermal growthfactor receptor variant III (EGFRvIII) (SEQ ID NO: 33) in a competitivebinding assay.

In certain embodiments, the anti-EGFR antibodies, or antigen bindingportions thereof, bind to an epitope within the amino acid sequenceCGADSYEMEEDGVRKC (SEQ ID NO: 45) and compete with Ab1 (or an anti-EGFRantibody comprises a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO: 1 and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO: 5) forbinding to epidermal growth factor receptor variant III (EGFRvIII) (SEQID NO: 33) in a competitive binding assay; and bind to EGFRvIII (SEQ IDNO: 33) with a K_(d) of about 8.2×10⁻⁹ M or less, as determined bysurface plasmon resonance.

In certain embodiments, the anti-EGFR antibodies, or antigen bindingportions thereof, bind to an epitope within the amino acid sequenceCGADSYEMEEDGVRKC (SEQ ID NO: 45) and compete with Ab1 (or an anti-EGFRantibody comprises a heavy chain variable domain comprising the aminoacid sequence set forth in SEQ ID NO: 1 and a light chain variabledomain comprising the amino acid sequence set forth in SEQ ID NO: 5) forbinding to epidermal growth factor receptor variant III (EGFRvIII) (SEQID NO: 33) in a competitive binding assay; and inhibit or decrease tumorgrowth in an in vivo xenograft mouse model. More specifically, theantibodies, or antigen binding portions thereof, of the invention areable to inhibit tumor growth by at least about 50% in an in vivo humannon-small-cell lung carcinoma (NSCLC) xenograft assay relative to ahuman IgG antibody which is not specific for EGFR when administered atthe same dose and dosing periodicity. Alternatively, the antibodies, orantigen binding portions thereof, of the invention are able to inhibitor decrease tumor growth in an in vivo human non-small-cell lungcarcinoma (NSCLC) xenograft assay relative to a human IgG antibody whichis not specific for EGFR by at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, or at leastabout 80%, when administered at the same dose and dosing periodicity. Incertain embodiments, the antibodies, or antigen-binding portionsthereof, of the invention are able to inhibit or decrease tumor growthin an in vivo human non-small-cell lung carcinoma (NSCLC) xenograftassay relative to a human IgG antibody which is not specific for EGFR byfrom about 80% to about 90%, or from about 84% to about 90%, or fromabout 88% to about 90%, when administered at the same dose and dosingperiodicity.

Antibodies having combinations of any of the aforementionedcharacteristics are contemplated as aspects of the invention. ADCs ofthe invention, described in more detail below, may also have any of theforegoing characteristics.

In one embodiment, the invention includes an anti-hEGFR antibody, orantigen binding portion thereof, comprising an LC CDR3 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 40, an LC CDR2 domaincomprising the amino acid sequence set forth in SEQ ID NO: 39, and an LCCDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:38; and an HC CDR3 domain comprising the amino acid sequence set forthin SEQ ID NO: 37, an HC CDR2 domain comprising the amino acid sequenceset forth in SEQ ID NO: 36, and an HC CDR1 domain comprising the aminoacid sequence set forth in SEQ ID NO: 35.

In one embodiment, the invention includes an anti-hEGFR antibody, orantigen binding portion thereof, comprising a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,and 78; and a light chain variable region comprising an amino acidsequence selected from the group consisting of 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, and 79.

In one embodiment, the invention includes an anti-hEGFR antibody, orantigen binding portion thereof, comprising an HC CDR set (CDR1, CDR2,and CDR3) selected from the group consisting of SEQ ID NOs: 10, 11, and12; SEQ ID NOs: 16, 17, and 18; SEQ ID NOs: 10, 11, and 19; SEQ ID NOs:20, 11, and 12; SEQ ID NOs: 21, 3, and 22; SEQ ID NOs: 16, 17, and 19;SEQ ID NOs: 2, 3, and 4; SEQ ID NOs: 10, 3, and 12; SEQ ID NOs: 80, 11,and 18; SEQ ID NOs: 80, 3, and 18; SEQ ID NOs: 20, 3, and 12; SEQ IDNOs: 80, 11, and 12; and SEQ ID NOs: 81, 11, and 22; and an LC lightchain CDR set (CDR1, CDR2, and CDR3) selected from the group consistingof SEQ ID NOs: 6, 7, and 8; SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 26,27, and 28; SEQ ID NOs: 29, 30, and 31; SEQ ID NOs: 6, 7, and 84; SEQ IDNOs: 82, 83, and 31; and SEQ ID NOs: 82, 27, and 85, wherein theantibody, or antigen binding portion thereof, does not comprise both theHC CDR set of SEQ ID NOs: 2, 3, and 4, and the LC CDR set of SEQ ID NOs:6, 7, and 8.

Preferably, anti-EGFR antibodies of the invention, exhibit a highcapacity to reduce or to neutralize EGFR activity, e.g., as assessed byany one of several in vitro and in vivo assays known in the art. Forexample, inhibition of phosphorylation of EGFR in an EGFR expressingcell line, e.g., the h292 cell line, can be measured. In certainembodiments, the isolated antibody, or antigen binding portion thereof,binds human EGFR, wherein the antibody, or antigen binding portionthereof, dissociates from human EGFR (EGFR 1-525) with a K_(D) rateconstant of about 5.9×10⁻⁷ M or less, as determined by surface plasmonresonance. Alternatively, the antibody, or an antigen binding portionthereof, may dissociate from human EGFR (1-525) with a K_(D) rateconstant of about 4.2×10⁻⁷ M, as determined by surface plasmonresonance. Alternatively, the antibody, or an antigen binding portionthereof, may dissociate from human EGFR (1-525) with a k_(off) rateconstant of about K_(D) rate constant of about 2.5×10⁻⁷ M, as determinedby surface plasmon resonance. In certain embodiments, the anti-EGFRantibodies, or antigen binding portion thereof, of the invention have aK_(D) rate constant of between 5.9×10⁻⁷ M and 5×10⁻⁹ M.

Alternatively, the antibody, or an antigen binding portion thereof, maydissociate from human EGFRvIII with a K_(D) rate constant of about6.1×10⁻⁹M or less, as determined by surface plasmon resonance.Alternatively, the antibody, or an antigen binding portion thereof, maydissociate from human EGFRvIII with a K_(D) rate constant of about3.9×10⁻⁹M or less, as determined by surface plasmon resonance.Alternatively, the antibody, or an antigen binding portion thereof, maydissociate from human EGFRvIII with a K_(D) rate constant of about2.3×10⁻⁹M or less, as determined by surface plasmon resonance.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is antibody AbA. AbA has improvedbinding affinity over Ab1 for EGFR, and also exhibits unique in vitroand in vivo characteristics relative to Ab1. AbA binds EGFR in an invitro keratinocyte binding assay with a much affinity greater than thatof Ab1. Further, AbA is able to inhibit or decrease tumor growth in axenograft H292 cell assay. Notably, AbA has improved in vitro and invivo characteristics which are comparable to other Ab1 variantantibodies which had higher binding affinity than that of AbA. Despitehaving lower binding affinity in comparison to other Ab1 variantantibodies (see for example AbP and AbQ vs. AbA in FIG. 3), AbA wascomparable at inhibiting cell growth in an in vivo assay.

The term “AbA” is meant to include an IgG antibody having at least thesix CDRs of AbA. The AbA antibody has the same light chain as that ofAb1, but has a heavy chain containing six amino acid sequence changesrelative to parental antibody Ab1 (four amino acid changes in thevariable region and two changes in the constant region of the heavychain). The AbA antibody comprises a heavy chain variable regioncomprising a CDR3 domain comprising the amino acid sequence of SEQ IDNO: 12, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:11, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:10, and a light chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 8, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 7, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 6. The heavy chainvariable region of AbA is defined by the amino acid sequence set forthin SEQ ID NO: 9, and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 5. The full length heavy chain of antibodyAbA is set forth in the amino acid sequence described in SEQ ID NO: 15,while the full length light chain of antibody AbA is set forth in theamino acid sequence described in SEQ ID NO: 13 (see FIG. 2). The nucleicacid sequence of the heavy chain of AbA is provided below:

(SEQ ID NO: 86) gaggtgcaactccaagagagcgggcccggcctcgtgaagccctctcagactctgtccctgacttgcactgtgagcgggtattccatcagcagagacttcgcatggaactggatccgccagcctcccggtaagggactggagtggatggggtacatcagctacaacggtaatacacgctatcagccctccctgaagtctcgcattaccattagtcgcgatacctccaagaaccagttctttctgaaactcaacagcgtgacagccgctgacaccgccacctactactgcgtgaccgccagcagggggttcccttactggggccagggcactctggtcaccgtttcttctgcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgcgaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaThe nucleic acid sequence of the light chain of AbA is provided below:

(SEQ ID NO: 87) Gacatccagatgacccagtccccctccagtatgtctgtgtctgtgggcgaccgtgtgaccattacctgccactcctcccaggacatcaatagcaatatcggttggttgcaacagaagccaggcaagtccttcaaagggctgatttaccatggtaccaacctggacgacggggttcctagtcgtttcagcggctccgggtccggaaccgattacactctgaccatcagcagtttgcagcctgaggactttgctacctattattgtgtgcagtacgctcagttcccatggactttcggcgggggcaccaaactggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbB. The AbBantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 17, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 16, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, the invention providesan antibody having a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 64 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 65.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbC. The AbCantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 84, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, the invention providesan antibody having a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 66 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 67.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbD. The AbDantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 31, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 83, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 82. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 68 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 69.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbE. The AbEantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 3, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 2, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 85, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 82. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 50 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 51.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbF. The AbFantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 10, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6. In further embodiments, the invention providesan antibody having a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 52 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 53.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbG. The AbGantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 17, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 16, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 25, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 24, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 23. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 72 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 73.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbH. The AbHantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 80, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 25, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 24, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 23. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 54 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 55.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbJ. The AbJantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 80, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 25, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 24, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 23. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 56 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 57.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbK. The AbKantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 19, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 10, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 26. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 74 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 75.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbL. The AbLantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 80, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 26. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 58 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 59.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbM. The AbMantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 20, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 26. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 76 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 77.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbN. The AbNantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 20, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 26. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 60 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 61.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbO. The AbOantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 80, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 27, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 26. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 62 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 63.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbP. The AbPantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 22, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 3, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 21, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 31, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 30, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 29. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 78 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 79.

In one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, which is the antibody AbQ. The AbQantibody comprises a heavy chain variable region comprising a CDR3domain comprising the amino acid sequence of SEQ ID NO: 22, a CDR2domain comprising the amino acid sequence of SEQ ID NO: 11, and a CDR1domain comprising the amino acid sequence of SEQ ID NO: 81, and a lightchain variable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 31, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 30, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 29. In further embodiments, the inventionprovides an antibody having a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 70 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 71.

As described in Table 1 in the examples set forth below, the Ab1 variantantibody sequences provide amino acid consensus sequences that representCDR domains resulting in improved binding to the Ab1 EGFR epitope. Thus,in one embodiment, the invention features an anti-EGFR antibody, orantigen binding portion thereof, comprising a light chain variableregion comprising a CDR3 domain comprising the amino acid sequence setforth as SEQ ID NO: 40, a CDR2 domain comprising the amino acid sequenceset forth as SEQ ID NO: 39, and a CDR1 domain comprising the amino acidsequence set forth as SEQ ID NO: 38; and a heavy chain variable regioncomprising a CDR3 domain comprising the amino acid sequence set forth asSEQ ID NO: 37, a CDR2 domain comprising the amino acid sequence setforth as SEQ ID NO: 36, and a CDR1 domain comprising the amino acidsequence set forth as SEQ ID NO: 35.

In one embodiment, the anti-Epidermal Growth Factor Receptor (anti-EGFR)antibody, or antigen binding portion thereof, comprises a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of 50, 52, 53, 56, 58, 60, 62, 64, 66, and 68; and alight chain variable region comprising an amino acid sequence selectedfrom the group consisting of 51, 53, 55, 57, 59, 61, 63, 65, 67, and 69.

In a further embodiment, the anti-EGFR antibody, or antigen bindingportion thereof, of the invention comprises a heavy chain variableregion comprising a CDR3 domain comprising an amino acid sequence as setforth in SEQ ID NO: 12, 18, 19, and 22; a CDR2 domain comprising anamino acid sequence as set forth in SEQ ID NO: 11 or 17; and a CDR1domain comprising an amino acid sequence as set forth in SEQ ID NO: 10,16, 20, and 21; and a light chain variable region comprising a CDR3domain comprising an amino acid sequence as set forth in SEQ ID NO: 8,25, 28, and 31; a CDR2 domain comprising an amino acid sequence as setforth in SEQ ID NO: 7, 24, 27, and 30; and a CDR1 domain comprising anamino acid sequence as set forth in SEQ ID NO: 6, 23, 26, and 29.

Phosphorylation and proliferation assays demonstrated that theantibodies described herein, inhibited EGFR mediated phosphorylation andtumor cell growth. For example, as set forth in Example 6, the EGFRantibodies (as tested) of the invention were shown to inhibit tumor cellgrowth in vivo.

The foregoing anti-EGFR antibody CDR sequences establish a novel familyof EGFR binding proteins, isolated in accordance with this invention,and comprising polypeptides that include the CDR sequences listed inTables 1 to 3 below.

To generate and to select CDRs having preferred EGFR binding and/orneutralizing activity with respect to hEGFR, standard methods known inthe art for generating antibodies, or antigen binding portions thereof,and assessing the EGFR binding and/or neutralizing characteristics ofthose antibodies, or antigen binding portions thereof, may be used,including but not limited to those specifically described herein.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgDconstant region. In certain embodiments, the anti-EGFR antibody, orantigen binding portion thereof, comprises a heavy chain immunoglobulinconstant domain selected from the group consisting of a human IgGconstant domain, a human IgM constant domain, a human IgE constantdomain, and a human IgA constant domain. In further embodiments, theantibody, or antigen binding portion thereof, has an IgG1 heavy chainconstant region, an IgG2 heavy chain constant region, an IgG3 constantregion, or an IgG4 heavy chain constant region. Preferably, the heavychain constant region is an IgG1 heavy chain constant region or an IgG4heavy chain constant region. Furthermore, the antibody can comprise alight chain constant region, either a kappa light chain constant regionor a lambda light chain constant region. Preferably, the antibodycomprises a kappa light chain constant region. Alternatively, theantibody portion can be, for example, a Fab fragment or a single chainFv fragment.

In certain embodiments, the anti-EGFR antibody binding portion is a Fab,a Fab′, a F(ab′)2, a Fv, a disulfide linked Fv, an scFv, a single domainantibody, or a diabody.

In certain embodiments, the anti-EGFR antibody, or antigen bindingportion thereof, is a multispecific antibody, e.g. a bispecificantibody.

In certain embodiments, the anti-EGFR antibody, or antigen bindingportion thereof, comprises a heavy chain constant region comprising theamino acid sequence set forth in SEQ ID NO: 41 and/or a light chainconstant region comprising the amino acid sequence set forth in SEQ IDNO: 43.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are have been described (Winter, et al. U.S. Pat. Nos.5,648,260 and 5,624,821, incorporated by reference herein). The Fcportion of an antibody mediates several important effector functionse.g. cytokine induction, ADCC, phagocytosis, complement dependentcytotoxicity (CDC) and half-life/clearance rate of antibody andantigen-antibody complexes. In some cases these effector functions aredesirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to FcγRs and complement C1q,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered.

One embodiment of the invention includes a recombinant chimeric antigenreceptor (CAR) comprising the binding regions of the antibodiesdescribed herein, e.g., the heavy and/or light chain CDRs of AbA. Arecombinant CAR, as described herein, may be used to redirect T cellspecificity to an antigen in a human leukocyte antigen (HLA)-independentfashion. Thus, CARS of the invention may be used in immunotherapy tohelp engineer a human subject's own immune cells to recognize and attackthe subject's tumor (see, e.g., U.S. Pat. Nos. 6,410,319; 8,389,282;8,822,647; 8,906,682; 8,911,993; 8,916,381; 8,975,071; and U.S. PatentAppln. Publ. No. US20140322275, each of which is incorporated byreference herein with respect to CAR technology). This type ofimmunotherapy is called adoptive cell transfer (ACT), and may be used totreat cancer in a subject in need thereof.

An anti-EGFR CAR of the invention preferably contains a extracellularantigen-binding domain specific for EGFR (e.g. EGFRvIII), atransmembrane domain which is used to anchor the CAR into a T cell, andone or more intracellular signaling domains. In one embodiment of theinvention, the CAR includes a transmembrane domain that comprises atransmembrane domain of a protein selected from the group consisting ofthe alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon,CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,CD134, CD137 and CD154. In one embodiment of the invention, the CARcomprises a costimulatory domain, e.g., a costimulatory domaincomprising a functional signaling domain of a protein selected from thegroup consisting of OX40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). In certain embodiments ofthe invention, the CAR comprises an scFv comprising the CDR or variableregions described herein e.g., CDRs or variable regions from the AbAantibody, a transmembrane domain, a costimulatory domain (e.g., afunctional signaling domain from CD28 or 4-1BB), and a signaling domaincomprising a functional signaling domain from CD3 (e.g., CD3-zeta).

In certain embodiments, the invention includes a T cell comprising a CAR(also referred to as a CAR T cell) comprising antigen binding regions,e.g. CDRs, of the antibodies described herein or an scFv describedherein.

In certain embodiments of the invention, the CAR comprises a variableheavy light chain comprising a CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 40, a CDR2 domain comprising the aminoacid sequence set forth in SEQ ID NO: 39, and a CDR1 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 38; and a heavy chainvariable region comprising a CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 37, a CDR2 domain comprising the aminoacid sequence set forth in SEQ ID NO: 36, and a CDR1 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 35.

In certain embodiments of the invention, the CAR comprises a variableheavy light chain comprising a CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 12, a CDR2 domain comprising the aminoacid sequence set forth in SEQ ID NO: 11, and a CDR1 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 10; and a heavy chainvariable region comprising a CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 8, a CDR2 domain comprising the aminoacid sequence set forth in SEQ ID NO: 7, and a CDR1 domain comprisingthe amino acid sequence set forth in SEQ ID NO: 6.

One embodiment of the invention includes a labeled anti-EGFR antibody,or antibody portion thereof, where the antibody is derivatized or linkedto one or more functional molecule(s) (e.g., another peptide orprotein). For example, a labeled antibody can be derived by functionallylinking an antibody or antibody portion of the invention (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a pharmaceuticalagent, a protein or peptide that can mediate the association of theantibody or antibody portion with another molecule (such as astreptavidin core region or a polyhistidine tag), and/or a cytotoxic ortherapeutic agent selected from the group consisting of a mitoticinhibitor, an antitumor antibiotic, an immunomodulating agent, a vectorfor gene therapy, an alkylating agent, an antiangiogenic agent, anantimetabolite, a boron-containing agent, a chemoprotective agent, ahormone, an antihormone agent, a corticosteroid, a photoactivetherapeutic agent, an oligonucleotide, a radionuclide agent, atopoisomerase inhibitor, a tyrosine kinase inhibitor, a radiosensitizer,and a combination thereof.

Useful detectable agents with which an antibody or antibody portionthereof, may be derivatized include fluorescent compounds. Exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

In one embodiment, the antibody of the invention is conjugated to animaging agent. Examples of imaging agents that may be used in thecompositions and methods described herein include, but are not limitedto, a radiolabel (e.g., indium), an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, a magnetic label, and biotin.

In one embodiment, the antibodies or ADCs are linked to a radiolabel,such as, but not limited to, indium (¹¹¹In). ¹¹¹Indium may be used tolabel the antibodies and ADCs described herein for use in identifyingEGFR positive tumors. In a certain embodiment, anti-EGFR antibodies (orADCs) described herein are labeled with via a bifunctional chelatorwhich is a bifunctional cyclohexyl diethylenetriaminepentaacetic acid(DTPA) chelate (see U.S. Pat. Nos. 5,124,471; 5,434,287; and 5,286,850,each of which is incorporated herein by reference).

Another embodiment of the invention provides a glycosylated bindingprotein wherein the anti-EGFR antibody or antigen binding portionthereof comprises one or more carbohydrate residues. Nascent in vivoprotein production may undergo further processing, known aspost-translational modification. In particular, sugar (glycosyl)residues may be added enzymatically, a process known as glycosylation.The resulting proteins bearing covalently linked oligosaccharide sidechains are known as glycosylated proteins or glycoproteins. Antibodiesare glycoproteins with one or more carbohydrate residues in the Fcdomain, as well as the variable domain. Carbohydrate residues in the Fcdomain have important effect on the effector function of the Fc domain,with minimal effect on antigen binding or half-life of the antibody (R.Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast,glycosylation of the variable domain may have an effect on the antigenbinding activity of the antibody. Glycosylation in the variable domainmay have a negative effect on antibody binding affinity, likely due tosteric hindrance (Co, M. S., et al., Mol. Immunol. (1993) 30:1361-1367),or result in increased affinity for the antigen (Wallick, S. C., et al.,Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBO J. (1991)10:2717-2723).

One aspect of the invention is directed to generating glycosylation sitemutants in which the O- or N-linked glycosylation site of the bindingprotein has been mutated. One skilled in the art can generate suchmutants using standard well-known technologies. Glycosylation sitemutants that retain the biological activity, but have increased ordecreased binding activity, are another object of the invention.

In still another embodiment, the glycosylation of the anti-EGFR antibodyor antigen binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.5,714,350 and 6,350,861, each of which is incorporated herein byreference in its entirety.

Additionally or alternatively, a modified anti-EGFR antibody of theinvention can be made that has an altered type of glycosylation, such asa hypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP1,176,195; PCT Publications WO 03/035835; WO 99/54342 80, each of whichis incorporated herein by reference in its entirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

Differing protein glycosylation may result in differing proteincharacteristics. For instance, the efficacy of a therapeutic proteinproduced in a microorganism host, such as yeast, and glycosylatedutilizing the yeast endogenous pathway may be reduced compared to thatof the same protein expressed in a mammalian cell, such as a CHO cellline. Such glycoproteins may also be immunogenic in humans and showreduced half-life in vivo after administration. Specific receptors inhumans and other animals may recognize specific glycosyl residues andpromote the rapid clearance of the protein from the bloodstream. Otheradverse effects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using recombinant techniques, apractitioner may generate antibodies or antigen binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (U.S. patent PublicationNos. 20040018590 and 20020137134 and PCT publication WO2005100584 A2).

Antibodies may be produced by any of a number of techniques. Forexample, expression from host cells, wherein expression vector(s)encoding the heavy and light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it ispossible to express antibodies in either prokaryotic or eukaryotic hostcells, expression of antibodies in eukaryotic cells is preferable, andmost preferable in mammalian host cells, because such eukaryotic cells(and in particular mammalian cells) are more likely than prokaryoticcells to assemble and secrete a properly folded and immunologicallyactive antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the invention.For example, it may be desirable to transfect a host cell with DNAencoding functional fragments of either the light chain and/or the heavychain of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some, or all, of the DNA encoding either or bothof the light and heavy chains that is not necessary for binding to theantigens of interest. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the antigens ofinterest by crosslinking an antibody of the invention to a secondantibody by standard chemical crosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr-CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked to CMVenhancer/AdMLP promoter regulatory elements to drive high levels oftranscription of the genes. The recombinant expression vector alsocarries a DHFR gene, which allows for selection of CHO cells that havebeen transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell in asuitable culture medium until a recombinant antibody is synthesized.Recombinant antibodies of the invention may be produced using nucleicacid molecules corresponding to the amino acid sequences disclosedherein. In one embodiment, the nucleic acid molecules set forth in SEQID NOs: 86 and/or 87 are used in the production of a recombinantantibody. The method can further comprise isolating the recombinantantibody from the culture medium.

III. Anti-EGFR Antibody Drug Conjugates (ADCs)

Anti-EGFR antibodies described herein may be conjugated to a drug moietyto form an anti-EGFR Antibody Drug Conjugate (ADC). Antibody-drugconjugates (ADCs) may increase the therapeutic efficacy of antibodies intreating disease, e.g., cancer, due to the ability of the ADC toselectively deliver one or more drug moiety(s) to target tissues, suchas a tumor-associated antigen, e.g., EGFR expressing tumors. Thus, incertain embodiments, the invention provides anti-EGFR ADCs fortherapeutic use, e.g., treatment of cancer.

Anti-EGFR ADCs of the invention comprise an anti-EGFR antibody, i.e., anantibody that specifically binds to EGFR, linked to one or more drugmoieties. The specificity of the ADC is defined by the specificity ofthe antibody, i.e., anti-EGFR. In one embodiment, an anti-EGFR antibodyis linked to one or more cytotoxic drug(s) which is delivered internallyto a transformed cancer cell expressing EGFR.

Examples of drugs that may be used in the anti-EGFR ADC of the inventionare provided below, as are linkers that may be used to conjugate theantibody and the one or more drug(s). The terms “drug,” “agent,” and“drug moiety” are used interchangeably herein. The terms “linked” and“conjugated” are also used interchangeably herein and indicate that theantibody and moiety are covalently linked.

In some embodiments, the ADC has the following formula (formula I):

Ab-(L-D)_(n)  (I)

wherein Ab is the antibody, e.g., anti-EGFR antibody AbA, and (L-D) is aLinker-Drug moiety. The Linker-Drug moiety is made of L- which is aLinker, and -D, which is a drug moiety having, for example, cytostatic,cytotoxic, or otherwise therapeutic activity against a target cell,e.g., a cell expressing EGFR; and n is an integer from 1 to 20. In someembodiments, n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, or is 1. The DAR of an ADC is equivalent to the “n” referredto in Formula I. In one embodiment, the ADC has a formula ofAb-(L-D)_(n), wherein Ab is an anti-EGFR antibody, e.g. AbA, L is alinker, e.g., valine citrulline (vc), D is a drug, e.g., an auristatinsuch as MMAF or MMAE, and n is 2 to 4 (equivalent to a DAR of 2-4).Additional details regarding drugs (D of Formula I) and linkers (L ofFormula I) that may be used in the ADCs of the invention, as well asalternative ADC structures, are described below.

A. Anti-EGFR ADCs: Exemplary Drugs for Conjugation

Anti-EGFR antibodies may be used in ADCs to target one or more drug(s)to a cell of interest, e.g., a cancer cell expressing EGFR. Theanti-EGFR ADCs of the invention provide a targeted therapy that may, forexample, reduce the side effects often seen with anti-cancer therapies,as the one or more drug(s) is delivered to a specific cell.

Auristatins

Anti-EGFR antibodies of the invention, e.g., the AbA antibody, may beconjugated to at least one auristatin. Auristatins represent a group ofdolastatin analogs that have generally been shown to possess anticanceractivity by interfering with microtubule dynamics and GTP hydrolysis,thereby inhibiting cellular division. For example, Auristatin E (U.S.Pat. No. 5,635,483) is a synthetic analogue of the marine naturalproduct dolastatin 10, a compound that inhibits tubulin polymerizationby binding to the same site on tubulin as the anticancer drugvincristine (G. R. Pettit, Prog. Chem. Org. Nat. Prod, 70: 1-79 (1997)).Dolastatin 10, auristatin PE, and auristatin E are linear peptideshaving four amino acids, three of which are unique to the dolastatinclass of compounds. Exemplary embodiments of the auristatin subclass ofmitotic inhibitors include, but are not limited to, monomethylauristatin D (MMAD or auristatin D derivative), monomethyl auristatin E(MMAE or auristatin E derivative), monomethyl auristatin F (MMAF orauristatin F derivative), auristatin F phenylenediamine (AFP),auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid-AEester (AEVB). The synthesis and structure of auristatin derivatives aredescribed in U.S. Patent Application Publication Nos. 2003-0083263,2005-0238649 and 2005-0009751; International Patent Publication No. WO04/010957, International Patent Publication No. WO 02/088172, and U.S.Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860;5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284;5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;4,879,278; 4,816,444; and 4,486,414, each of which is incorporated byreference herein.

In one embodiment, anti-EGFR antibodies of the invention, e.g., AbA, areconjugated to at least one MMAE (mono-methyl auristatin E). Monomethylauristatin E (MMAE, vedotin) inhibits cell division by blocking thepolymerization of tubulin. Because of its super toxicity, it also cannotbe used as a drug itself. In recent cancer therapy developments, it islinked to a monoclonal antibody (mAb) that recognizes a specific markerexpression in cancer cells and directs MMAE to the cancer cells. In oneembodiment, the linker linking MMAE to the anti-EGFR antibody is stablein extracellular fluid (i.e., the medium or environment that is externalto cells), but is cleaved by cathepsin once the ADC has bound to thespecific cancer cell antigen and entered the cancer cell, thus releasingthe toxic MMAE and activating the potent anti-mitotic mechanism.

In one embodiment, an anti-EGFR antibody described herein, e.g., AbA, isconjugated to at least one MMAF (monomethylauristatin F). Monomethylauristatin F (MMAF) inhibits cell division by blocking thepolymerization of tubulin. It has a charged C-terminal phenylalanineresidue that attenuates its cytotoxic activity compared to its unchargedcounterpart MMAE. Because of its super toxicity, it cannot be used as adrug itself, but can be linked to a monoclonal antibody (mAb) thatdirects it to the cancer cells. In one embodiment, the linker to theanti-EGFR antibody is stable in extracellular fluid, but is cleaved bycathepsin once the conjugate has entered a tumor cell, thus activatingthe anti-mitotic mechanism.

The structures of MMAF and MMAE are provided below.

An example of AbA-vcMMAE is also provided in FIG. 11. Notably, FIG. 11describes a situation where the antibody (e.g., AbA) is coupled to asingle drug and, therefore, has a DAR of 1. In certain embodiments, theADC will have a DAR of 2 to 8, or, alternatively, 2 to 4.

Other Drugs for Conjugation

Examples of drugs that may be used in ADCs, i.e., drugs that may beconjugated to the anti-EGFR antibodies of the invention, are providedbelow, and include mitotic inhibitors, antitumor antibiotics,immunomodulating agents, gene therapy vectors, alkylating agents,antiangiogenic agents, antimetabolites, boron-containing agents,chemoprotective agents, hormone agents, glucocorticoids, photoactivetherapeutic agents, oligonucleotides, radioactive isotopes,radiosensitizers, topoisomerase inhibitors, tyrosine kinase inhibitors,and combinations thereof.

1. Mitotic Inhibitors

In one aspect, anti-EGFR antibodies may be conjugated to one or moremitotic inhibitor(s) to form an ADC for the treatment of cancer. Theterm “mitotic inhibitor”, as used herein, refers to a cytotoxic and/ortherapeutic agent that blocks mitosis or cell division, a biologicalprocess particularly important to cancer cells. A mitotic inhibitordisrupts microtubules such that cell division is prevented, often byaffecting microtubule polymerization or microtubule depolymerization.Thus, in one embodiment, an anti-EGFR antibody of the invention isconjugated to one or more mitotic inhibitor(s) that disrupts microtubuleformation by inhibiting tubulin polymerization. In one embodiment, themitotic inhibitor used in the ADCs of the invention is Ixempra(ixabepilone). Examples of mitotic inhibitors that may be used in theanti-EGFR ADCs of the invention are provided below. Included in thegenus of mitotic inhibitors are auristatins, described above.

a. Dolastatins

The anti-EGFR antibodies of the invention may be conjugated to at leastone dolastatin to form an ADC. Dolastatins are short peptidic compoundsisolated from the Indian Ocean sea hare Dolabella auricularia (seePettit et al., J. Am. Chem. Soc., 1976, 98, 4677). Examples ofdolastatins include dolastatin 10 and dolatstin 15. Dolastatin 15, aseven-subunit depsipeptide derived from Dolabella auricularia, and is apotent antimitotic agent structurally related to the antitubulin agentdolastatin 10, a five-subunit peptide obtained from the same organism.Thus, in one embodiment, the anti-EGFR ADC of the invention comprises ananti-EGFR antibody, as described herein, and at least one dolastatin.Auristatins, described above, are synthetic derivatives of dolastatin10.

b. Maytansinoids

The anti-EGFR antibodies of the invention may be conjugated to at leastone maytansinoid to form an ADC. Maytansinoids are potent antitumoragents that were originally isolated from members of the higher plantfamilies Celastraceae, Rhamnaceae and Euphorbiaceae, as well as somespecies of mosses (Kupchan et al, J. Am. Chem. Soc. 94:1354-1356 [1972];Wani et al, J. Chem. Soc. Chem. Commun. 390: [1973]; Powell et al, J.Nat. Prod. 46:660-666 [1983]; Sakai et al, J. Nat. Prod. 51:845-850[1988]; and Suwanborirux et al, Experientia 46:117-120 [1990]). Evidencesuggests that maytansinoids inhibit mitosis by inhibiting polymerizationof the microtubule protein tubulin, thereby preventing formation ofmicrotubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al.,Science, 189, 1002-1005 (1975)). Maytansinoids have been shown toinhibit tumor cell growth in vitro using cell culture models, and invivo using laboratory animal systems. Moreover, the cytotoxicity ofmaytansinoids is 1,000-fold greater than conventional chemotherapeuticagents, such as, for example, methotrexate, daunorubicin, andvincristine (see, e.g., U.S. Pat. No. 5,208,020).

Maytansinoids to include maytansine, maytansinol, C-3 esters ofmaytansinol, and other maytansinol analogues and derivatives (see, e.g.,U.S. Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated byreference herein). C-3 esters of maytansinol can be naturally occurringor synthetically derived. Moreover, both naturally occurring andsynthetic C-3 maytansinol esters can be classified as a C-3 ester withsimple carboxylic acids, or a C-3 ester with derivatives ofN-methyl-L-alanine, the latter being more cytotoxic than the former.Synthetic maytansinoid analogues are described in, for example, Kupchanet al., J. Med. Chem., 21, 31-37 (1978).

Suitable maytansinoids for use in ADCs of the invention can be isolatedfrom natural sources, synthetically produced, or semi-syntheticallyproduced. Moreover, the maytansinoid can be modified in any suitablemanner, so long as sufficient cytotoxicity is preserved in the ultimateconjugate molecule. In this regard, maytansinoids lack suitablefunctional groups to which antibodies can be linked. A linking moietydesirably is utilized to link the maytansinoid to the antibody to formthe conjugate, and is described in more detail in section III.B. Thestructure of an exemplary maytansinoid, mertansine (DM1), is providedbelow.

Representative examples of maytansinoids include, but are not limited,to DM1 (N²′-deacetyl-N²′-(3-mercapto-1-oxopropyl)-maytansine; alsoreferred to as mertansine, drug maytansinoid 1; ImmunoGen, Inc.; seealso Chari et al. (1992) Cancer Res 52:127), DM2, DM3(N²′-deacetyl-N²′-(4-mercapto-1-oxopentyl)-maytansine), DM4(4-methyl-4-mercapto-1-oxopentyl)-maytansine) and maytansinol (asynthetic maytansinoid analog). Other examples of maytansinoids aredescribed in U.S. Pat. No. 8,142,784, incorporated by reference herein.

Ansamitocins are a group of maytansinoid antibiotics that have beenisolated from various bacterial sources. These compounds have potentantitumor activities. Representative examples include, but are notlimited to ansamitocin P1, ansamitocin P2, ansamitocin P3, andansamitocin P4.

In one embodiment of the invention, an anti-EGFR antibody is conjugatedto at least one DM1. In one embodiment, an anti-EGFR antibody isconjugated to at least one DM2. In one embodiment, an anti-EGFR antibodyis conjugated to at least one DM3. In one embodiment, an anti-EGFRantibody is conjugated to at least one DM4.

d. Plant Alkaloids

The anti-EGFR antibodies of the invention may be conjugated to at leastone plant alkaloid, e.g., a taxane or vinca alkaloid. Plant alkaloidsare chemotherapy treatments derived made from certain types of plants.The vinca alkaloids are made from the periwinkle plant (catharanthusrosea), whereas the taxanes are made from the bark of the Pacific Yewtree (taxus). Both the vinca alkaloids and taxanes are also known asantimicrotubule agents, and are described in more detail below.

Taxanes

Anti-EGFR antibodies described herein may be conjugated to at least onetaxane. The term “taxane” as used herein refers to the class ofantineoplastic agents having a mechanism of microtubule action andhaving a structure that includes the taxane ring structure and astereospecific side chain that is required for cytostatic activity. Alsoincluded within the term “taxane” are a variety of known derivatives,including both hydrophilic derivatives, and hydrophobic derivatives.Taxane derivatives include, but not limited to, galactose and mannosederivatives described in International Patent Application No. WO99/18113; piperazino and other derivatives described in WO 99/14209;taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Pat.No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfenamidederivatives described in U.S. Pat. No. 5,821,263; and taxol derivativedescribed in U.S. Pat. No. 5,415,869, each of which is incorporated byreference herein. Taxane compounds have also previously been describedin U.S. Pat. Nos. 5,641,803, 5,665,671, 5,380,751, 5,728,687, 5,415,869,5,407,683, 5,399,363, 5,424,073, 5,157,049, 5,773,464, 5,821,263,5,840,929, 4,814,470, 5,438,072, 5,403,858, 4,960,790, 5,433,364,4,942,184, 5,362,831, 5,705,503, and 5,278,324, all of which areexpressly incorporated by reference. Further examples of taxanesinclude, but are not limited to, docetaxel (Taxotere; Sanofi Aventis),paclitaxel (Abraxane or Taxol; Abraxis Oncology), and nanoparticlepaclitaxel (ABI-007/Abraxene; Abraxis Bioscience).

In one embodiment, the anti-EGFR antibody of the invention is conjugatedto at least one docetaxel. In one embodiment, the anti-EGFR antibody ofthe invention is conjugated to at least one paclitaxel.

Vinca Alkaloids

In one embodiment, the anti-EGFR antibody is conjugated to at least onevinca alkaloid. Vinca alkaloids are a class of cell-cycle-specific drugsthat work by inhibiting the ability of cancer cells to divide by actingupon tubulin and preventing the formation of microtubules. Examples ofvinca alkaloids that may be used in the ADCs of the invention include,but are not limited to, vindesine sulfate, vincristine, vinblastine andvinorelbine.

2. Antitumor Antibiotics

Anti-EGFR antibodies of the invention may be conjugated to one or moreantitumor antibiotic(s) for the treatment of cancer. As used herein, theterm “antitumor antibiotic” means an antineoplastic drug that blockscell growth by interfering with DNA and is made from a microorganism.Often, antitumor antibiotics either break up DNA strands or slow down orstop DNA synthesis. Examples of antitumor antibiotics that may beincluded in the anti-EGFR ADCs of the invention include, but are notlimited to, actinomycines (e.g., pyrrolo[2,1-c][1,4]benzodiazepines),anthracyclines, calicheamicins, and duocarmycins, described in moredetail below.

a. Actinomycines

The anti-EGFR antibodies of the invention may be conjugated to at leastone actinomycine. Actinomycines are a subclass of antitumor antibioticsisolated from bacteria of the genus Streptomyces. Representativeexamples actinomycines include, but are not limited to, actinomycin D(Cosmegen [also known as actinomycin, dactinomycin, actinomycin IV,actinomycin C1], Lundbeck, Inc.), anthramycin, chicamycin A, DC-81,mazethramycin, neothramycin A, neothramycin B, porothramycin,prothracarcin B, SG2285, sibanomicin, sibiromycin and tomaymycin. In oneembodiment, the anti-EGFR antibody of the invention is conjugated to atleast one pyrrolobenzodiazepine (PBD). Examples of PBDs include, but arenot limited to, anthramycin, chicamycin A, DC-81, mazethramycin,neothramycin A, neothramycin B, porothramycin, prothracarcin B, SG2000(SJG-136), SG2202 (ZC-207), SG2285 (ZC-423), sibanomicin, sibiromycinand tomaymycin. Thus, in one embodiment, anti-EGFR antibodies of theinvention are conjugated to at least one actinomycine, e.g., actinomycinD, or at least one PBD, e.g., a pyrrolobenzodiazepine (PBD) dimer.

The structures of PBDs can be found, for example, in U.S. PatentApplication Pub. Nos. 2013/0028917 and 2013/0028919, and in WO2011/130598 A1, each of which are incorporated herein by reference intheir entirety. The generic structure of a PBD is provided below.

PBDs differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring, there is generally an imine(N═C), a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position which is the electrophilic centreresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11α position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. The PBD examples provided herein may be conjugated to theanti-EGFR antibodies of the invention. Further examples of PBDs whichmay be conjugated to the anti-EGFR antibodies of the invention can befound, for example, in U.S. Patent Application Publication Nos.2013/0028917 A1 and 2013/0028919 A1, in U.S. Pat. No. 7,741,319 B2, andin WO 2011/130598 A1 and WO 2006/111759 A1, each of which areincorporated herein by reference in their entirety.

A representative PBD dimer having the following formula II may beconjugated to the anti-EGFR antibodies of the invention:

wherein:

R² is of formula III:

where A is a C₅₋₇ aryl group, X is a group conjugated to the Linker unitselected from the group consisting of —O—, —S—, —C(O)O—, —C(O)—,—NH(C═O)—, and —N(R^(N))—, wherein R^(N) is selected from the groupconsisting of H, C₁₋₄ alkyl and (C₂H₄O)_(m)CH₃, where m is 1 to 3, andeither:

(i) Q¹ is a single bond, and Q² is selected from the group consisting ofa single bond and —Z—(CH₂)_(n)—, where Z is selected from the groupconsisting of a single bond, O, S and NH and n is from 1 to 3; or

-   -   (ii) Q¹ is —C—H═CH—, and Q² is a single bond;

R¹² is a C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group consisting of halo, nitro, cyano,C₁₋₁₂ alkoxy, C₃₋₂₀ heterocycloalkoxy, C₅₋₂₀ aryloxy, heteroaryloxy,alkylalkoxy, arylalkoxy, alkylaryloxy, heteroarylalkoxy,alkylheteroaryloxy, C₁₋₇alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

R⁶ and R⁹ are independently selected from the group consisting of H, R,OH, OR, SH, SR, NH₂, NHR, NRR′, nitro, Me₃Sn and halo;

-   -   where R and R′ are independently selected from the group        consisting of optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀        heterocyclyl and C₅₋₂₀ aryl groups;

R⁷ is selected from the group consisting of H, R, OH, OR, SH, SR, NH₂,NHR, NHRR′, nitro, Me₃Sn and halo;

either:

(a) R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl:

(b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogenand carbon atoms to which they are bound; or

(c) R¹⁰ is H and R¹¹ is SO_(z)M, where z is 2 or 3;

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, selected from the group consisting of O, S, NH, and anaromatic ring;

Y and Y′ are is selected from the group consisting of O, S, and NH;

R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ andR⁹ respectively and R^(10′) and R^(11′) are the same as R¹⁰ and R¹¹, andeach M is a monovalent pharmaceutically acceptable cation or both Mgroups together are a divalent pharmaceutically acceptable cation.

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl”includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussedbelow.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇); O₁: oxirane(C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran)(C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆),oxepin (C₇); S₁: thiirane (C₃), thietane (C₄), thiolane(tetrahydrothiophene) (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane(C₇); O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇); O₃: trioxane(C₆); N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅),imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆); N₁S₁: thiazoline (C₅), thiazolidine(C₅), thiomorpholine (C₆); N₂O₁: oxadiazine (C₆); O₁S₁: oxathiole (C₅)and oxathiane (thioxane) (C₆); and, N₁O₁5₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein,pertains to an aryl group having 5 or 6 ring atoms.

In one embodiment, the anti-EGFR antibodies of the invention may beconjugated to a PBD dimer having the following formula:

wherein the above structure describes the PBD dimer SG2202 (ZC-207) andis conjugated to the anti-EGFR antibody of the invention via a linker L.SG2202 (ZC-207) is disclosed in, for example, U.S. Patent App. Pub. No.2007/0173497, which is incorporated herein by reference in its entirety.

In another embodiment, a PBD dimer, SGD-1882, is conjugated to anti-EGFRantibody of the invention via a drug linker, as depicted in FIG. 21.SGD-1882 is disclosed in Sutherland et al. (2013) Blood 122(8):1455 andin U.S Patent App. Pub. No. 2013/0028919, which is incorporated hereinbe reference in its entirety. As described in FIG. 21, the PBD dimerSGD-1882 may be conjugated to an antibody via an mc-val-ala-dipeptidelinker (collectively referred to as SGD-1910 in FIG. 21). In a certainembodiment, an anti-EGFR antibody, as disclosed herein, is conjugated tothe PBD dimer described in FIG. 21. Thus, in a further embodiment, theinvention includes an anti-EGFR antibody, as disclosed herein,conjugated to a PBD dimer via a mc-val-ala-dipeptide linker, asdescribed in FIG. 21.

In certain embodiments, the invention includes an anti-EGFR antibodycomprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 12, a CDR2 domaincomprising the amino acid sequence of SEQ ID NO: 11, and a CDR1 domaincomprising the amino acid sequence of SEQ ID NO: 10, and a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 8, a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 7, and a CDR1 domain comprising the amino acidsequence of SEQ ID NO: 6, conjugated to a PBD, including, but notlimited to, the PBD dimer described in FIG. 21. In certain embodiments,the invention includes an anti-EGFR antibody comprising the heavy chainvariable region of AbA as defined by the amino acid sequence set forthin SEQ ID NO: 9, and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 5, wherein the antibody is conjugated to aPBD, such as, but not limited to, the exemplary PBD dimer of FIG. 21.

b. Anthracyclines

Anti-EGFR antibodies of the invention may be conjugated to at least oneanthracycline. Anthracyclines are a subclass of antitumor antibioticsisolated from bacteria of the genus Streptomyces. Representativeexamples include, but are not limited to daunorubicin (Cerubidine,Bedford Laboratories), doxorubicin (Adriamycin, Bedford Laboratories;also referred to as doxorubicin hydrochloride, hydroxydaunorubicin, andRubex), epirubicin (Ellence, Pfizer), and idarubicin (Idamycin; PfizerInc.). Thus, in one embodiment, the anti-EGFR antibody of the inventionis conjugated to at least one anthracycline, e.g., doxorubicin.

c. Calicheamicins

The anti-EGFR antibodies of the invention may be conjugated to at leastone calicheamicin. Calicheamicins are a family of enediyne antibioticsderived from the soil organism Micromonospora echinospora.Calicheamicins bind the minor groove of DNA and induce double-strandedDNA breaks, resulting in cell death with a 100 fold increase over otherchemotherapeutics (Damle et al. (2003) Curr Opin Pharmacol 3:386).Preparation of calicheamicins that may be used as drug conjugates in theinvention have been described, see U.S. Pat. Nos. 5,712,374; 5,714,586;5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296.Structural analogues of calicheamicin which may be used include, but arenot limited to, γ₁ ¹, α₂ ¹, α₃ ¹, N-acetyl-γ₁ ¹, PSAG and θ¹ ₁ (Hinmanet al., Cancer Research 53:3336-3342 (1993), Lode et al., CancerResearch 58:2925-2928 (1998) and the aforementioned U.S. Pat. Nos.5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710;5,773,001; and 5,877,296). Thus, in one embodiment, the anti-EGFRantibody of the invention is conjugated to at least one calicheamicin.

d. Duocarmycins

Anti-EGFR antibodies of the invention may be conjugated to at least oneduocarmycin. Duocarmycins are a subclass of antitumor antibioticsisolated from bacteria of the genus Streptomyces. (see Nagamura andSaito (1998) Chemistry of Heterocyclic Compounds, Vol. 34, No. 12).Duocarmycins bind to the minor groove of DNA and alkylate the nucleobaseadenine at the N3 position (Boger (1993) Pure and Appl Chem 65(6):1123;and Boger and Johnson (1995) PNAS USA 92:3642). Synthetic analogs ofduocarmycins include, but are not limited to, adozelesin, bizelesin, andcarzelesin. Thus, in one embodiment, the anti-EGFR antibody of theinvention is conjugated to at least one duocarmycin.

e. Other Antitumor Antibiotics

In addition to the foregoing, additional antitumor antibiotics that maybe used in the anti-EGFR ADCs of the invention include bleomycin(Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also knownas mithramycin).

3. Immunomodulating Agents

In one aspect, anti-EGFR antibodies of the invention may be conjugatedto at least one immunomodulating agent. As used herein, the term“immunomodulating agent” refers to an agent that can stimulate or modifyan immune response. In one embodiment, an immunomodulating agent is animmunostimuator which enhances a subject's immune response. In anotherembodiment, an immunomodulating agent is an immunosuppressant whichprevents or decreases a subject's immune response. An immunomodulatingagent may modulate myeloid cells (monocytes, macrophages, dendriticcells, megakaryocytes and granulocytes) or lymphoid cells (T cells, Bcells and natural killer (NK) cells) and any further differentiated cellthereof. Representative examples include, but are not limited to,bacillus calmette-guerin (BCG) and levamisole (Ergamisol). Otherexamples of immunomodulating agents that may be used in the ADCs of theinvention include, but are not limited to, cancer vaccines, cytokines,and immunomodulating gene therapy.

a. Cancer Vaccines

Anti-EGFR antibodies of the invention may be conjugated to a cancervaccine. As used herein, the term “cancer vaccine” refers to acomposition (e.g., a tumor antigen and a cytokine) that elicits atumor-specific immune response. The response is elicited from thesubject's own immune system by administering the cancer vaccine, or, inthe case of the instant invention, administering an ADC comprising ananti-EGFR antibody and a cancer vaccine. In preferred embodiments, theimmune response results in the eradication of tumor cells in the body(e.g., primary or metastatic tumor cells). The use of cancer vaccinesgenerally involves the administration of a particular antigen or groupof antigens that are, for example, present on the surface a particularcancer cell, or present on the surface of a particular infectious agentshown to facilitate cancer formation. In some embodiments, the use ofcancer vaccines is for prophylactic purposes, while in otherembodiments, the use is for therapeutic purposes. Non-limiting examplesof cancer vaccines that may be used in the anti-EGFR ADCs of theinvention include, recombinant bivalent human papillomavirus (HPV)vaccine types 16 and 18 vaccine (Cervarix, GlaxoSmithKline), recombinantquadrivalent human papillomavirus (HPV) types 6, 11, 16, and 18 vaccine(Gardasil, Merck & Company), and sipuleucel-T (Provenge, Dendreon).Thus, in one embodiment, the anti-EGFR antibody of the invention isconjugated to at least one cancer vaccine that is either animmunostimulator or is an immunosuppressant.

b. Cytokines

The anti-EGFR antibodies of the invention may be conjugated to at leastone cytokine. The term “cytokine” generally refers to proteins releasedby one cell population which act on another cell as intercellularmediators. Cytokines directly stimulate immune effector cells andstromal cells at the tumor site and enhance tumor cell recognition bycytotoxic effector cells (Lee and Margolin (2011) Cancers 3:3856).Numerous animal tumor model studies have demonstrated that cytokineshave broad anti-tumor activity and this has been translated into anumber of cytokine-based approaches for cancer therapy (Lee and Margoli,supra). Recent years have seen a number of cytokines, including GM-CSF,IL-7, IL-12, IL-15, IL-18 and IL-21, enter clinical trials for patientswith advanced cancer (Lee and Margoli, supra).

Examples of cytokines that may be used in the ADCs of the inventioninclude, but are not limited to, parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF; platelet-growthfactor; transforming growth factors (TGFs); insulin-like growth factor-Iand -II; erythropoietin (EPO); osteoinductive factors; interferons suchas interferon α, β, and γ, colony stimulating factors (CSFs);granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factor; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines. Thus, in one embodiment, the inventionprovides an ADC comprising an anti-EGFR antibody described herein and acytokine.

c. Colony-Stimulating Factors (CSFs)

The anti-EGFR antibodies of the invention may be conjugated to at leastone colony stimulating factor (CSF). Colony stimulating factors (CSFs)are growth factors that assist the bone marrow in making red bloodcells. Because some cancer treatments (e.g., chemotherapy) can affectwhite blood cells (which help fight infection), colony-stimulatingfactors may be introduced to help support white blood cell levels andstrengthen the immune system. Colony-stimulating factors may also beused following a bone marrow transplant to help the new marrow startproducing white blood cells. Representative examples of CSFs that may beused in the anti-EGFR ADCs of the invention include, but are not limitedto erythropoietin (Epoetin), filgrastim (Neopogen (also known asgranulocyte colony-stimulating factor (G-CSF); Amgen, Inc.),sargramostim (leukine (granulocyte-macrophage colony-stimulating factorand GM-CSF); Genzyme Corporation), promegapoietin, and Oprelvekin(recombinant IL-11; Pfizer, Inc.). Thus, in one embodiment, theinvention provides an ADC comprising an anti-EGFR antibody describedherein and a CSF.

4. Gene Therapy

The anti-EGFR antibody of the invention may be conjugated to at leastone nucleic acid (directly or indirectly via a carrier) for genetherapy. Gene therapy generally refers to the introduction of geneticmaterial into a cell whereby the genetic material is designed to treat adisease. As it pertains to immunomoduatory agents, gene therapy is usedto stimulate a subject's natural ability to inhibit cancer cellproliferation or kill cancer cells. In one embodiment, the anti-EGFR ADCof the invention comprises a nucleic acid encoding a functional,therapeutic gene that is used to replace a mutated or otherwisedysfuntional (e.g. truncated) gene associated with cancer. In otherembodiments, the anti-EGFR ADC of the invention comprises a nucleic acidthat encodes for or otherwise provides for the production of atherapeutic protein to treat cancer. The nucleic acid that encodes thetherapeutic gene may be directly conjugated to the anti-EGFR antibody,or alternatively, may be conjugated to the anti-EGFR antibody through acarrier. Examples of carriers that may be used to deliver a nucleic acidfor gene therapy include, but are not limited to, viral vectors orliposomes.

5. Alkylating Agents

The anti-EGFR antibodies of the invention may be conjugated to one ormore alkylating agent(s). Alkylating agents are a class ofantineoplastic compounds that attaches an alkyl group to DNA. Examplesof alkylating agents that may be used in the ADCs of the inventioninclude, but are not limited to, alkyl sulfonates, ethylenimimes,methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas,triazines and hydrazines.

a. Alkyl Sulfonates

The anti-EGFR antibodies of the invention may be conjugated to at leastone alkyl sulfonate. Alkyl sulfonates are a subclass of alkylatingagents with a general formula: R—SO₂—O—R¹, wherein R and R¹ aretypically alkyl or aryl groups. A representative example of an alkylsulfonate includes, but is not limited to, busulfan (Myleran,GlaxoSmithKline; Busulfex IV, PDL BioPharma, Inc.).

b. Nitrogen Mustards

The anti-EGFR antibodies of the invention may be conjugated to at leastone nitrogen mustard. Representative examples of this subclass ofanti-cancer compounds include, but are not limited to chlorambucil(Leukeran, GlaxoSmithKline), cyclophosphamide (Cytoxan, Bristol-MyersSquibb; Neosar, Pfizer, Inc.), estramustine (estramustine phosphatesodium or Estracyt), Pfizer, Inc.), ifosfamide (Ifex, Bristol-MyersSquibb), mechlorethamine (Mustargen, Lundbeck Inc.), and melphalan(Alkeran or L-Pam or phenylalanine mustard; GlaxoSmithKline).

c. Nitrosoureas

The anti-EGFR antibody of the invention may be conjugated to at leastone nitrosourea. Nitrosoureas are a subclass of alkylating agents thatare lipid soluble. Representative examples include, but are not limitedto, carmustine (BCNU [also known as BiCNU,N,N-Bis(2-chloroethyl)-N-nitrosourea, or1,3-bis(2-chloroethyl)-l-nitrosourea], Bristol-Myers Squibb),fotemustine (also known as Muphoran), lomustine (CCNU or1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea, Bristol-Myers Squibb),nimustine (also known as ACNU), and streptozocin (Zanosar, TevaPharmaceuticals).

d. Triazines and Hydrazines

The anti-EGFR antibody of the invention may be conjugated to at leastone triazine or hydrazine. Triazines and hydrazines are a subclass ofnitrogen-containing alkylating agents. In some embodiments, thesecompounds spontaneously decompose or can be metabolized to produce alkyldiazonium intermediates that facilitate the transfer of an alkyl groupto nucleic acids, peptides, and/or polypeptides, thereby causingmutagenic, carcinogenic, or cytotoxic effects. Representative examplesinclude, but are not limited to dacarbazine (DTIC-Dome, Bayer HealthcarePharmaceuticals Inc.), procarbazine (Mutalane, Sigma-TauPharmaceuticals, Inc.), and temozolomide (Temodar, Schering Plough).

e. Other Alkylating Agents

The anti-EGFR antibodies of the invention may be conjugated to at leastone ethylenimine, methylamine derivative, or epoxide. Ethylenimines area subclass of alkylating agents that typically containing at least oneaziridine ring. Epoxides represent a subclass of alkylating agents thatare characterized as cyclic ethers with only three ring atoms.

Representatives examples of ethylenimines include, but are not limitedto thiopeta (Thioplex, Amgen), diaziquone (also known as aziridinylbenzoquinone (AZQ)), and mitomycin C. Mitomycin C is a natural productthat contains an aziridine ring and appears to induce cytoxicity throughcross-linking DNA (Dorn R T, et al. Cancer Res. 1985; 45:3510; Kennedy KA, et al Cancer Res. 1985; 45:3541).

Representative examples of methylamine derivatives and their analogsinclude, but are not limited to, altretamine (Hexalen, MGI Pharma,Inc.), which is also known as hexamethylamine and hexastat.Representative examples of epoxides of this class of anti-cancercompound include, but are not limited to dianhydrogalactitol.Dianhydrogalactitol (1,2:5,6-dianhydrodulcitol) is chemically related tothe aziridines and generally facilitate the transfer of an alkyl groupthrough a similar mechanism as described above. Dibromodulcitol ishydrolyzed to dianhydrogalactitol and thus is a pro-drug to an epoxide(Sellei C, et al. Cancer Chemother Rep. 1969; 53:377).

6. Antiangiogenic Agents

In one aspect, the anti-EGFR antibodies described herein are conjugatedto at least one antiangiogenic agent. Antiangiogenic agents inhibit thegrowth of new blood vessels. Antiangiogenic agents exert their effectsin a variety of ways. In some embodiments, these agents interfere withthe ability of a growth factor to reach its target. For example,vascular endothelial growth factor (VEGF) is one of the primary proteinsinvolved in initiating angiogenesis by binding to particular receptorson a cell surface. Thus, certain antiangiogenic agents, that prevent theinteraction of VEGF with its cognate receptor, prevent VEGF frominitiating angiogenesis. In other embodiments, these agents interferewith intracellular signaling cascades. For example, once a particularreceptor on a cell surface has been triggered, a cascade of otherchemical signals is initiated to promote the growth of blood vessels.Thus, certain enzymes, for example, some tyrosine kinases, that areknown to facilitate intracellular signaling cascades that contribute to,for example, cell proliferation, are targets for cancer treatment. Inother embodiments, these agents interfere with intercellular signalingcascades. Yet, in other embodiments, these agents disable specifictargets that activate and promote cell growth or by directly interferingwith the growth of blood vessel cells. Angiogenesis inhibitoryproperties have been discovered in more than 300 substances withnumerous direct and indirect inhibitory effects.

Representative examples of antiangiogenic agents that may be used in theADCs of the invention include, but are not limited to, angiostatin, ABXEGF, C1-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900,CP358, PD153035, AG1478, IMC-C225 (Erbitux, ZD1839 (Iressa), OSI-774,Erlotinib (tarceva), angiostatin, arrestin, endostatin, BAY 12-9566 andw/fluorouracil or doxorubicin, canstatin, carboxyamidotriozole and withpaclitaxel, EMD121974, S-24, vitaxin, dimethylxanthenone acetic acid,IM862, Interleukin-12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMab,angiozyme (ribozyme), IMC-1C11, Neovastat, marimstat, prinomastat,BMS-275291, COL-3, MM1270, SU101, SU6668, SU11248, SU5416, withpaclitaxel, with gemcitabine and cisplatin, and with irinotecan andcisplatin and with radiation, tecogalan, temozolomide and PEG interferonα2b, tetrathiomolybdate, TNP-470, thalidomide, CC-5013 and withtaxotere, tumstatin, 2-methoxyestradiol, VEGF trap, mTOR inhibitors(deforolimus, everolimus (Afinitor, Novartis PharmaceuticalCorporation), and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinaseinhibitors (e.g., erlotinib (Tarceva, Genentech, Inc.), imatinib(Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa,AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb),sunitinib (Sutent, Pfizer, Inc.), nilotinib (Tasigna, NovartisPharmaceutical Corporation), lapatinib (Tykerb, GlaxoSmithKlinePharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide3-kinases (PI3K).

7. Antimetabolites

The anti-EGFR antibodies of the invention may be conjugated to at leastone antimetabolite. Antimetabolites are types of chemotherapy treatmentsthat are very similar to normal substances within the cell. When thecells incorporate an antimetabolite into the cellular metabolism, theresult is negative for the cell, e.g., the cell is unable to divide.Antimetabolites are classified according to the substances with whichthey interfere. Examples of antimetabolies that may be used in the ADCsof the invention include, but are not limited to, a folic acidantagonist (e.g., methotrexate), a pyrimidine antagonist (e.g.,5-Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine),a purine antagonist (e.g., 6-Mercaptopurine and 6-Thioguanine) and anadenosine deaminase inhibitor (e.g., Cladribine, Fludarabine, Nelarabineand Pentostatin), as described in more detail below.

a. Antifolates

The anti-EGFR antibodies of the invention may be conjugated to at leastone antifolate. Antifolates are a subclass of antimetabolites that arestructurally similar to folate. Representative examples include, but arenot limited to, methotrexate, 4-amino-folic acid (also known asaminopterin and 4-aminopteroic acid), lometrexol (LMTX), pemetrexed(Alimpta, Eli Lilly and Company), and trimetrexate (Neutrexin, Ben VenueLaboratories, Inc.)

b. Purine Antagonists

The anti-EGFR antibodies of the invention may be conjugated to at leastone purine antagonist. Purine analogs are a subclass of antimetabolitesthat are structurally similar to the group of compounds known aspurines. Representative examples of purine antagonists include, but arenot limited to, azathioprine (Azasan, Salix; Imuran, GlaxoSmithKline),cladribine (Leustatin [also known as 2-CdA], Janssen Biotech, Inc.),mercaptopurine (Purinethol [also known as 6-mercaptoethanol],GlaxoSmithKline), fludarabine (Fludara, Genzyme Corporation),pentostatin (Nipent, also known as 2′-deoxycoformycin (DCF)),6-thioguanine (Lanvis [also known as thioguanine], GlaxoSmithKline).

c. Pyrimidine Antagonists

The anti-EGFR antibodies of the invention may be conjugated to at leastone pyrimidine antagonist. Pyrimidine antagonists are a subclass ofantimetabolites that are structurally similar to the group of compoundsknown as purines. Representative examples of pyrimidine antagonistsinclude, but are not limited to azacitidine (Vidaza, CelgeneCorporation), capecitabine (Xeloda, Roche Laboratories), Cytarabine(also known as cytosine arabinoside and arabinosylcytosine, BedfordLaboratories), decitabine (Dacogen, Eisai Pharmaceuticals),5-fluorouracil (Adrucil, Teva Pharmaceuticals; Efudex, ValeantPharmaceuticals, Inc), 5-fluoro-2′-deoxyuridine 5′-phosphate (FdUMP),5-fluorouridine triphosphate, and gemcitabine (Gemzar, Eli Lilly andCompany).

8. Boron-Containing Agents

The anti-EGFR antibody of the invention may be conjugated to at leastone boron containing agent. Boron-containing agents comprise a class ofcancer therapeutic compounds which interfere with cell proliferation.Representative examples of boron containing agents include, but are notlimited, to borophycin and bortezomib (Velcade, MilleniumPharmaceuticals).

9. Chemoprotective Agents

The anti-EGFR antibodies of the invention may be conjugated to at leastone chemoprotective agent. Chemoprotective drugs are a class ofcompounds, which help protect the body against specific toxic effects ofchemotherapy. Chemoprotective agents may be administered with variouschemotherapies in order to protect healthy cells from the toxic effectsof chemotherapy drugs, while simultaneously allowing the cancer cells tobe treated with the administered chemotherapeutic. Representativechemoprotective agents include, but are not limited to amifostine(Ethyol, Medimmune, Inc.), which is used to reduce renal toxicityassociated with cumulative doses of cisplatin, dexrazoxane (Totect,Apricus Pharma; Zinecard), for the treatment of extravasation caused bythe administration of anthracycline (Totect), and for the treatment ofcardiac-related complications caused by the administration of theantitumor antibiotic doxorubicin (Zinecard), and mesna (Mesnex,Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitisduring chemotherapy treatment with ifocfamide.

10. Hormone agents

The anti-EGFR antibody of the invention may be conjugated to at leastone hormone agent. A hormone agent (including synthetic hormones) is acompound that interferes with the production or activity of endogenouslyproduced hormones of the endocrine system. In some embodiments, thesecompounds interfere with cell growth or produce a cytotoxic effect.Non-limiting examples include androgens, estrogens, medroxyprogesteroneacetate (Provera, Pfizer, Inc.), and progestins.

11. Antihormone Agents

The anti-EGFR antibodies of the invention may be conjugated to at leastone antihormone agent. An “antihormone” agent is an agent thatsuppresses the production of and/or prevents the function of certainendogenous hormones. In one embodiment, the antihormone agent interfereswith the activity of a hormone selected from the group comprisingandrogens, estrogens, progesterone, and goanadotropin-releasing hormone,thereby interfering with the growth of various cancer cells.Representative examples of antihormone agents include, but are notlimited to, aminoglutethimide, anastrozole (Arimidex, AstraZenecaPharmaceuticals), bicalutamide (Casodex, AstraZeneca Pharmaceuticals),cyproterone acetate (Cyprostat, Bayer PLC), degarelix (Firmagon, FerringPharmaceuticals), exemestane (Aromasin, Pfizer Inc.), flutamide(Drogenil, Schering-Plough Ltd), fulvestrant (Faslodex, AstraZenecaPharmaceuticals), goserelin (Zolodex, AstraZeneca Pharmaceuticals),letrozole (Femara, Novartis Pharmaceuticals Corporation), leuprolide(Prostap), lupron, medroxyprogesterone acetate (Provera, Pfizer Inc.),Megestrol acetate (Megace, Bristol-Myers Squibb Company), tamoxifen(Nolvadex, AstraZeneca Pharmaceuticals), and triptorelin (Decapetyl,Ferring).

12. Corticosteroids

The anti-EGFR antibodies of the invention may be conjugated to at leastone corticosteroid. Corticosteroids may be used in the ADCs of theinvention to decrease inflammation. An example of a corticosteroidincludes, but is not limited to, a glucocorticoid, for example,prednisone (Deltasone, Pharmacia & Upjohn Company, a division of Pfizer,Inc.).

13. Photoactive Therapeutic Agents

The anti-EGFR antibodies of the invention may be conjugated to at leastone photoactive therapeutic agent. Photoactive therapeutic agentsinclude compounds that can be deployed to kill treated cells uponexposure to electromagnetic radiation of a particular wavelength.Therapeutically relevant compounds absorb electromagnetic radiation atwavelengths which penetrate tissue. In preferred embodiments, thecompound is administered in a non-toxic form that is capable ofproducing a photochemical effect that is toxic to cells or tissue uponsufficient activation. In other preferred embodiments, these compoundsare retained by cancerous tissue and are readily cleared from normaltissues. Non-limiting examples include various chromagens and dyes.

14. Oligonucleotides

The anti-EGFR antibodies of the invention may be conjugated to at leastone oligonucleotide. Oligonucleotides are made of short nucleic acidchains that work by interfering with the processing of geneticinformation. In some embodiments, the oligonucleotides for use in ADCsare unmodified single-stranded and/or double-stranded DNA or RNAmolecules, while in other embodiments, these therapeuticoligonucleotides are chemically-modified single-stranded and/ordouble-stranded DNA or RNA molecules. In one embodiment, theoligonulceotides used in the ADCs are relatively short (19-25nucleotides) and hybridize to a unique nucleic acid sequence in thetotal pool of nucleic acid targets present in cells. Some of theimportant oligonucleotide technologies include the antisenseoligonucleotides (including RNA interference (RNAi)), aptamers, CpGoligonucleotides, and ribozymes.

a. Antisense oligonucleotides

The anti-EGFR antibody of the invention may be conjugated to at leastone antisense oligonucleotide. Antisense oligonucleotides are designedto bind to RNA through Watson-Crick hybridization. In some embodimentsthe antisense oligonucleotide is complementary to a nucleotide encodinga region, domain, portion, or segment of EGFR. In some embodiments, theantisense oligonucleotide comprises from about 5 to about 100nucleotides, from about 10 to about 50 nucleotides, from about 12 toabout 35, and from about 18 to about 25 nucleotides. In someembodiments, the oligonucleotide is at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or at least 100% homologous to aregion, portion, domain, or segment of the EGFR gene. In someembodiments there is substantial sequence homology over at least 15, 20,25, 30, 35, 40, 50, or 100 consecutive nucleotides of the EGFR gene. Inpreferred embodiments, the size of these antisense oligonucleotidesranges from 12 to 25 nucleotides in length, with the majority ofantisense oligonucleotides being 18 to 21 nucleotides in length. Thereare multiple mechanisms that can be exploited to inhibit the function ofthe RNA once the oligonucleotide binds to the target RNA (Crooke S T.(1999). Biochim. Biophys. Acta, 1489, 30-42). The best-characterizedantisense mechanism results in cleavage of the targeted RNA byendogenous cellular nucleases, such as RNase H or the nucleaseassociated with the RNA interference mechanism. However,oligonucleotides that inhibit expression of the target gene bynon-catalytic mechanisms, such as modulation of splicing or translationarrest, can also be potent and selective modulators of gene function.

Another RNase-dependent antisense mechanism that has recently receivedmuch attention is RNAi (Fire et al. (1998). Nature, 391, 806-811; ZamoreP D. (2002). Science, 296, 1265-1269.). RNA interference (RNAi) is apost-transcriptional process where a double stranded RNA inhibits geneexpression in a sequence specific fashion. In some embodiments, the RNAieffect is achieved through the introduction of relatively longerdouble-stranded RNA (dsRNA), while in preferred embodiments, this RNAieffect is achieved by the introduction of shorter double-stranded RNAs,e.g. small interfering RNA (siRNA) and/or microRNA (miRNA). In yetanother embodiment, RNAi can also be achieved by introducing of plasmidthat generate dsRNA complementary to target gene. In each of theforegoing embodiments, the double-stranded RNA is designed to interferewith the gene expression of a particular the target sequence withincells. Generally, the mechanism involves conversion of dsRNA into shortRNAs that direct ribonucleases to homologous mRNA targets (summarized,Ruvkun, Science 2294:797 (2001)), which then degrades the correspondingendogenous mRNA, thereby resulting in the modulation of gene expression.Notably, dsRNA has been reported to have anti-proliferative properties,which makes it possible also to envisage therapeutic applications (Aubelet al., Proc. Natl. Acad. Sci., USA 88:906 (1991)). For example,synthetic dsRNA has been shown to inhibit tumor growth in mice (Levy etal. Proc. Nat. Acad. Sci. USA, 62:357-361 (1969)), is active in thetreatment of leukemic mice (Zeleznick et al., Proc. Soc. Exp. Biol. Med.130:126-128 (1969)), and inhibits chemically induced tumorigenesis inmouse skin (Gelboin et al., Science 167:205-207 (1970)). Thus, in apreferred embodiment, the invention provides for the use of antisenseoligonucleotides in ADCs for the treatment of breast cancer. In otherembodiments, the invention provides compositions and methods forinitiating antisense oligonucleotide treatment, wherein dsRNA interfereswith target cell expression of EGFR at the mRNA level. dsRNA, as usedabove, refers to naturally-occurring RNA, partially purified RNA,recombinantly produced RNA, synthetic RNA, as well as altered RNA thatdiffers from naturally-occurring RNA by the inclusion of non-standardnucleotides, non-nucleotide material, nucleotide analogs (e.g. lockednucleic acid (LNA)), deoxyribonucleotides, and any combination thereof.RNA of the invention need only be sufficiently similar to natural RNAthat it has the ability to mediate the antisense oligonucleotide-basedmodulation described herein.

b. Aptamers

The anti-EGFR antibodies of the invention may be conjugated to at leastone aptamer. An aptamer is a nucleic acid molecule that has beenselected from random pools based on its ability to bind other molecules.Like antibodies, aptamers can bind target molecules with extraordinaryaffinity and specificity. In many embodiments, aptamers assume complex,sequence-dependent, three-dimensional shapes that allow them to interactwith a target protein, resulting in a tightly bound complex analogous toan antibody-antigen interaction, thereby interfering with the functionof said protein. The particular capacity of aptamers to bind tightly andspecifically to their target protein underlines their potential astargeted molecular therapies.

c. CpG Oligonucleotides

The anti-EGFR antibodies of the invention may be conjugated to at leastone CpG oligonucleotide. Bacterial and viral DNA are known to be astrong activators of both the innate and specific immunity in humans.These immunologic characteristics have been associated with unmethylatedCpG dinucleotide motifs found in bacterial DNA. Owing to the fact thatthese motifs are rare in humans, the human immune system has evolved theability to recognize these motifs as an early indication of infectionand subsequently initiate immune responses. Therefore, oligonucleotidescontaining this CpG motif can be exploited to initiate an antitumorimmune response.

d. Ribozymes

The anti-EGFR antibody of the invention may be conjugated to at leastone ribozyme. Ribozymes are catalytic RNA molecules ranging from about40 to 155 nucleotides in length. The ability of ribozymes to recognizeand cut specific RNA molecules makes them potential candidates fortherapeutics. A representative example includes angiozyme.

15. Radionuclide Agents (Radioactive Isotopes)

The anti-EGFR antibodies of the invention may be conjugated to at leastone radionuclide agent. Radionuclide agents comprise agents that arecharacterized by an unstable nucleus that is capable of undergoingradioactive decay. The basis for successful radionuclide treatmentdepends on sufficient concentration and prolonged retention of theradionuclide by the cancer cell. Other factors to consider include theradionuclide half-life, the energy of the emitted particles, and themaximum range that the emitted particle can travel. In preferredembodiments, the therapeutic agent is a radionuclide selected from thegroup consisting of ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se,⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er; ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, an ²¹¹Pb. Also preferred are radionuclides that substantiallydecay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m,Tc-99m, Rh-103m, Pt-109, In-111 1, Sb-119, I-125, Ho-161, Os-189m andIr-192. Decay energies of useful beta-particle-emitting nuclides arepreferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1,Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵0, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³H, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb and the like.

16. Radiosensitizers

The anti-EGFR antibodies of the invention may be conjugated to at leastone radiosensitizer. The term “radiosensitizer,” as used herein, isdefined as a molecule, preferably a low molecular weight molecule,administered to animals in therapeutically effective amounts to increasethe sensitivity of the cells to be radiosensitized to electromagneticradiation and/or to promote the treatment of diseases that are treatablewith electromagnetic radiation. Radiosensitizers are agents that makecancer cells more sensitive to radiation therapy, while typically havingmuch less of an effect on normal cells. Thus, the radiosensitizer can beused in combination with a radiolabeled antibody or ADC. The addition ofthe radiosensitizer can result in enhanced efficacy when compared totreatment with the radiolabeled antibody or antibody fragment alone.Radiosensitizers are described in D. M. Goldberg (ed.), Cancer Therapywith Radiolabeled Antibodies, CRC Press (1995). Examples ofradiosensitizers include gemcitabine, 5-fluorouracil, taxane, andcisplatin.

Radiosensitizers may be activated by the electromagnetic radiation ofX-rays. Representative examples of X-ray activated radiosensitizersinclude, but are not limited to, the following: metronidazole,misonidazole, desmethylmisonidazole, pimonidazole, etanidazole,nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide,5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.Alternatively, radiosensitizers may be activated using photodynamictherapy (PDT). Representative examples of photodynamic radiosensitizersinclude, but are not limited to, hematoporphyrin derivatives,Photofrin(r), benzoporphyrin derivatives, NPe6, tin etioporphyrin(SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines,phthalocyanines, zinc phthalocyanine, and therapeutically effectiveanalogs and derivatives of the same.

16. Topoisomerase Inhibitors

The anti-EGFR antibodies of the invention may be conjugated to at leastone topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapyagents designed to interfere with the action of topoisomerase enzymes(topoisomerase I and II), which are enzymes that control the changes inDNA structure by catalyzing then breaking and rejoining of thephosphodiester backbone of DNA strands during the normal cell cycle.Representative examples of DNA topoisomerase I inhibitors include, butare not limited to, camptothecins and its derivatives irinotecan(CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin,GlaxoSmithKline Pharmaceuticals). Representative examples of DNAtopoisomerase II inhibitors include, but are not limited to, amsacrine,daunorubicin, doxotrubicin, epipodophyllotoxins, ellipticines,epirubicin, etoposide, razoxane, and teniposide.

17. Tyrosine Kinase Inhibitors

The anti-EGFR antibodies of the invention may be conjugated to at leastone tyrosine kinase inhibitor. Tyrosine kinases are enzymes within thecell that function to attach phosphate groups to the amino acidtyrosine. By blocking the ability of protein tyrosine kinases tofunction, tumor growth may be inhibited. Examples of tyrosine kinasesthat may be used on the ADCs of the invention include, but are notlimited to, Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib,Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Semaxanib,Sunitinib, and Vandetanib.

18. Other Agents

Examples of other agents that may be used in the ADCs of the inventioninclude, but are not limited to, abrin (e.g. abrin A chain), alphatoxin, Aleurites fordii proteins, amatoxin, crotin, curcin, dianthinproteins, diptheria toxin (e.g. diphtheria A chain and nonbinding activefragments of diphtheria toxin), deoxyribonuclease (Dnase), gelonin,mitogellin, modeccin A chain, momordica charantia inhibitor, neomycin,onconase, phenomycin, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), pokeweed antiviral protein, Pseudomonas endotoxin, Pseudomonasexotoxin (e.g. exotoxin A chain (from Pseudomonas aeruginosa)),restrictocin, ricin A chain, ribonuclease (Rnase), sapaonariaofficinalis inhibitor, saporin, alpha-sarcin, Staphylcoccalenterotoxin-A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin(Eloxatin, Sanofi Aventis), proteasome inhibitors (e.g. PS-341[bortezomib or Velcade]), HDAC inhibitors (vorinostat (Zolinza, Merck &Company, Inc.)), belinostat, entinostat, mocetinostat, andpanobinostat), COX-2 inhibitors, substituted ureas, heat shock proteininhibitors (e.g. Geldanamycin and its numerous analogs), adrenocorticalsuppressants, and the tricothecenes. (See, for example, WO 93/21232).Other agents also include asparaginase (Espar, Lundbeck Inc.),hydroxyurea, levamisole, mitotane (Lysodren, Bristol-Myers Squibb), andtretinoin (Renova, Valeant Pharmaceuticals Inc.).

It should be noted that the aforementioned groups of drug moieties thatmay be used in the anti-EGFR ADCs of the invention are not exclusive, inthat certain examples of drugs may be found in more than one category,e.g., ansamitocins are both mitotic inhibitors and antitumorantibiotics.

All stereoisomers of the above drug moieties are contemplated for thecompounds of the invention, i.e. any combination of R and Sconfigurations at the chiral carbons of D.

The above agents (i.e., naked agents not conjugated to an antibody) mayalso be used in combination therapies with the anti-EGFR antibodiesdescribed herein. In one embodiment, anti-EGFR antibodies or ADCs areused with any of the foregoing agents in a combination therapy to treatcancer, where the agent is administered prior to, at the same time as,or following administration of the anti-EGFR antibody or ADC to thesubject.

B. Anti-EGFR ADCs: Exemplary Linkers

An anti-EGFR ADC comprises an anti-EGFR antibody and at least onedrug(s), whereby the antibody and the at least one drug are conjugatedby a linker. The term “linker,” as used herein, refers to a chemicalmoiety that may be bifunctional or multifunctional, and is used toattach an antibody to a drug moiety. A linker may include oneconjugating component or may include multiple components. For example,the linker may include a spacer, which is a moiety that extends the druglinkage to avoid, for example, shielding the active site of the antibodyor improving the solubility of the ADC. Other examples of components oflinkers include a stretcher unit and an amino acid unit.

Two methods are commonly used for conjugating drugs to antibodies:alkylation of reduced interchain cysteine disulfides through anenzymatically non-cleavable maleimido or simple and cleavable disulfidelinker, and acylation of lysines by cleavable linear amino acids.

In one aspect, a linker covalently attaches an antibody to a drugmoiety. An ADC is prepared using a linker having reactive functionalityfor binding to the antibody and the drug. For example, a cysteine thiol,or an amine. e.g., N-terminus or amino acid side chain such as lysine,of the antibody may form a bond with a functional group of the linker.

In one aspect, a linker has a functionality that is capable of reactingwith a free cysteine present on an antibody to form a covalent bond.Nonlimiting exemplary such reactive functionalities include maleimide,haloacetamides, α-haloacetyl, activated esters such as succinimideesters, 4-nitrophenyl esters, pentafluorophenyl esters,tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonylchlorides, isocyanates, and isothiocyanates. See, e.g., the conjugationmethod at page 766 of Klussman, et al (2004), Bioconjugate Chemistry15(4):765-773.

In some embodiments, a linker has a functionality that is capable ofreacting with an electrophilic group present on an antibody. Exemplarysuch electrophilic groups include, but are not limited to, aldehyde andketone carbonyl groups. In some embodiments, a heteroatom of thereactive functionality of the linker can react with an electrophilicgroup on an antibody and form a covalent bond to an antibody unit.Nonlimiting exemplary such reactive functionalities include, but are notlimited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide.

Exemplary linker components include 6-maleimidocaproyl,maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”),alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”),N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“MCC”).

In one aspect, an anti-EGFR antibody is conjugated to an auristatin,e.g., MMAE, via a linker comprising maleimidocaproyl (“mc”), valinecitrulline (val-cit or “vc”), and PABA (referred to as a “mc-vc-PABAlinker”). Maleimidocaproyl acts as a linker to the anti-EGFR antibodyand is not cleavable. Val-cit is a dipeptide that is an amino acid unitof the linker and allows for cleavage of the linker by a protease,specifically the protease cathepsin B. Thus, the val-cit component ofthe linker provides a means for releasing the auristatin from the ADCupon exposure to the intracellular environment. Within the linker,p-aminobenzylalcohol (PABA) acts as a spacer and is self immolative,allowing for the release of the MMAE. The structure of themc-vc-PABA-MMAE linker is provided in FIG. 11.

Suitable linkers include, for example, cleavable and non-cleavablelinkers. A linker may be a “cleavable linker,” facilitating release of adrug. Nonlimiting exemplary cleavable linkers include acid-labilelinkers (e.g., comprising hydrazone), protease-sensitive (e.g.,peptidase-sensitive) linkers, photolabile linkers, ordisulfide-containing linkers (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020). A cleavable linker is typicallysusceptible to cleavage under intracellular conditions. Suitablecleavable linkers include, for example, a peptide linker cleavable by anintracellular protease, such as lysosomal protease or an endosomalprotease. In exemplary embodiments, the linker can be a dipeptidelinker, such as a valine-citrulline (val-cit) or a phenylalanine-lysine(phe-lys) linker.

Linkers are preferably stable extracellularly in a sufficient manner tobe therapeutically effective. Before transport or delivery into a cell,the ADC is preferably stable and remains intact, i.e. the antibodyremains conjugated to the drug moiety. Linkers that are stable outsidethe target cell may be cleaved at some efficacious rate once inside thecell. Thus, an effective linker will: (i) maintain the specific bindingproperties of the antibody; (ii) allow delivery, e.g., intracellulardelivery, of the drug moiety; and (iii) maintain the therapeutic effect,e.g., cytotoxic effect, of a drug moiety.

In one embodiment, the linker is cleavable under intracellularconditions, such that cleavage of the linker sufficiently releases thedrug from the antibody in the intracellular environment to betherapeutically effective. In some embodiments, the cleavable linker ispH-sensitive, i.e., sensitive to hydrolysis at certain pH values.Typically, the pH-sensitive linker is hydrolyzable under acidicconditions. For example, an acid-labile linker that is hydrolyzable inthe lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone,cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used.(See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchikand Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989,Biol. Chem. 264:14653-14661.) Such linkers are relatively stable underneutral pH conditions, such as those in the blood, but are unstable atbelow pH 5.5 or 5.0, the approximate pH of the lysosome. In certainembodiments, the hydrolyzable linker is a thioether linker (such as,e.g., a thioether attached to the therapeutic agent via an acylhydrazonebond (see, e.g., U.S. Pat. No. 5,622,929).

In other embodiments, the linker is cleavable under reducing conditions(e.g., a disulfide linker). A variety of disulfide linkers are known inthe art, including, for example, those that can be formed using SATA(N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.).

In some embodiments, the linker is cleavable by a cleaving agent, e.g.,an enzyme, that is present in the intracellular environment (e.g.,within a lysosome or endosome or caveolea). The linker can be, e.g., apeptidyl linker that is cleaved by an intracellular peptidase orprotease enzyme, including, but not limited to, a lysosomal or endosomalprotease. In some embodiments, the peptidyl linker is at least two aminoacids long or at least three amino acids long. Cleaving agents caninclude cathepsins B and D and plasmin, all of which are known tohydrolyze dipeptide drug derivatives resulting in the release of activedrug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm.Therapeutics 83:67-123). Most typical are peptidyl linkers that arecleavable by enzymes that are present in EGFR-expressing cells. Examplesof such linkers are described, e.g., in U.S. Pat. No. 6,214,345,incorporated herein by reference in its entirety and for all purposes.In a specific embodiment, the peptidyl linker cleavable by anintracellular protease is a Val-Cit linker or a Phe-Lys linker (see,e.g., U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the val-cit linker). One advantage of usingintracellular proteolytic release of the therapeutic agent is that theagent is typically attenuated when conjugated and the serum stabilitiesof the conjugates are typically high.

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released, for example, by antibody degradation. See U.S. PublicationNo. 20050238649 incorporated by reference herein in its entirety. An ADCcomprising a non-cleavable linker may be designed such that the ADCremains substantially outside the cell and interacts with certainreceptors on a target cell surface such that the binding of the ADCinitiates (or prevents) a particular cellular signaling pathway.

In some embodiments, the linker is substantially hydrophilic linker(e.g., PEG4Mal and sulfo-SPDB). A hydrophilic linker may be used toreduce the extent to which the drug may be pumped out of resistantcancer cells through MDR (multiple drug resistance) or functionallysimilar transporters.

In other embodiments, upon cleavage, the linker functions to directly orindirectly inhibit cell growth and/or cell proliferation. For example,in some embodiments, the linker, upon cleavage, can function as anintercalating agent, thereby inhibiting macromolecular biosynthesis(e.g. DNA replication, RNA transcription, and/or protein synthesis).

In other embodiments, the linker is designed to facilitate bystanderkilling (the killing of neighboring cells) through diffusion of thelinker-drug and/or the drug alone to neighboring cells. In other,embodiments, the linker promotes cellular internalization.

The presence of a sterically hindered disulfide can increase thestability of a particular disulfide bond, enhancing the potency of theADC. Thus, in one embodiment, the linker includes a sterically hindereddisulfide linkage. A sterically hindered disulfide refers to a disulfidebond present within a particular molecular environment, wherein theenvironment is characterized by a particular spatial arrangement ororientation of atoms, typically within the same molecule or compound,which prevents or at least partially inhibits the reduction of thedisulfide bond. Thus, the presence of bulky (or sterically hindering)chemical moieties and/or bulky amino acid side chains proximal to thedisulfide bond prevents or at least partially inhibits the disulfidebond from potential interactions that would result in the reduction ofthe disulfide bond.

Notably, the aforementioned linker types are not mutually exclusive. Forexample, in one embodiment, the linker used in the anti-EGFR ADCsdescribed herein is a non-cleavable linker that promotes cellularinternalization.

In some embodiments, the ADC has the following formula (formula I):

Ab-(L-D)_(n)  (I)

or a pharmaceutically acceptable salt or solvate thereof; wherein Ab isthe antibody, e.g., anti-EGFR antibody AbA, and (L-D) is a Linker-Drugmoiety. The Linker-Drug moiety is made of L- which is a Linker, and -D,which is a drug moiety having, for example, cytostatic, cytotoxic, orotherwise therapeutic activity against a target cell, e.g., a cellexpressing EGFR; and n is an integer from 1 to 20.

In some embodiments, n ranges from 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to4, 1 to 3, 1 to 2, or is 1.

In some embodiments, the -D moieties are the same. In yet anotherembodiment, the -D moieties are different.

As described above, the linker may be a single moiety or may include twoor more components. As such, in some embodiments, the ADC has thefollowing formula (II):

Ab-(A_(a)-W_(w)—Y_(y)-D)_(n)  (II)

or a pharmaceutically acceptable salt or solvate thereof, wherein Ab isthe antibody, e.g., anti-EGFR antibody AbA, and -A_(a)-W_(w)—Y_(y)— is aLinker (L) comprising three or more components, including -A-, which isis an optional Stretcher unit, a is 0 or 1, each —W— is independently anAmino Acid unit (or in some embodiments, a Glucuronide unit, See also USPublication No. 2012/0107332 A1), w is an integer ranging from 0 to 12,—Y— is a self-immolative spacer unit, y is 0, 1 or 2; -D is a drugmoiety having, for example, cytostatic, cytotoxic, or otherwisetherapeutic activity against the target cell, e.g., cell expressingEGFR; and n is an integer from 1 to 20.

In some embodiments, a linker component comprises a “stretcher unit” (A)that links an antibody to another linker component or to a drug moiety.Nonlimiting exemplary stretcher units are shown below (wherein the wavyline indicates sites of covalent attachment to an antibody, drug, oradditional linker components):

The stretcher unit (A), when present, is capable of linking an antibodyto an amino acid unit (—W—), if present, to a spacer unit (—Y—), ifpresent; or to a drug (-D) (see Formula II). Useful functional groupsthat can be present on the anti-EGFR antibodies described herein, eithernaturally or via chemical manipulation include, but are not limited to,sulfhydryl, amino, hydroxyl, the anomeric hydroxyl group of acarbohydrate, and carboxyl. Suitable functional groups are sulfhydryland amino. In one example, sulfhydryl groups can be generated byreduction of the intramolecular disulfide bonds of an anti-EGFRantibody. In another embodiment, sulfhydryl groups can be generated byreaction of an amino group of a lysine moiety of an anti-EGFR antibodywith 2-iminothiolane (Traut's reagent) or other sulfhydryl generatingreagents. In certain embodiments, the anti-EGFR antibody is arecombinant antibody and is engineered to carry one or more lysinemoieties. In certain other embodiments, the recombinant anti-EGFRantibody is engineered to carry additional sulfhydryl groups, e.g.,additional cysteines.

In one embodiment, the stretcher unit forms a bond with a sulfur atom ofthe antibody. The sulfur atom can be derived from a sulfhydryl group ofan antibody. Representative stretcher units of this embodiment aredepicted within the square brackets of Formulas Ma and Mb as shownbelow,

wherein L-, —W—, —Y—, -D, w and y are as defined above, and R′⁷ isselected from —C₁-C₁₀ alkylene-, —C₁-C₁₀ alkenylene-, —C₁-C₁₀alkynylene-, carbocyclo-, —O—(C₁-C₈ alkylene)-, O—(C₁-C₈ alkenylene)-,—O—(C₁-C₈ alkynylene)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, —C₂-C₁₀alkenylene-arylene, —C₂-C₁₀ alkynylene-arylene, arylene-C₁-C₁₀alkylene-, -arylene-C₂-C₁₀ alkenylene-, -arylene-C₂-C₁₀ alkynylene-,—C₁-C₁₀ alkylene-(carbocyclo)-, —C₂-C₁₀ alkenylene-(carbocyclo)-, C₂-C₁₀alkynylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-,-(carbocyclo)-C₂-C₁₀ alkenylene-, -(carbocyclo)-C₂-C₁₀ alkynylene,-heterocyclo-, —C₁-C₁₀ alkylene-(heterocyclo)-, —C₂-C₁₀alkenylene-(heterocyclo)-, —C₂-C₁₀ alkynylene-(heterocyclo)-,-(heterocyclo)-C₁-C₁₀ alkylene-, -(heterocyclo)-C₂-C₁₀ alkenylene-,-(heterocyclo)-C₁-C₁₀ alkynylene-, —(CH₂CH₂O)_(r)—, or—(CH₂CH₂O)_(r)—CH₂—, and r is an integer ranging from 1-10, wherein saidalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,carbocycle, carbocyclo, heterocyclo, and arylene radicals, whether aloneor as part of another group, are optionally substituted. In someembodiments, said alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkynyklene, aryl, carbocyle, carbocyclo, heterocyclo, and aryleneradicals, whether alone or as part of another group, are unsubstituted.In some embodiments, R¹⁷ is selected from —C₁-C₁₀ alkylene-,-carbocyclo-, —O—(C₁-C₈ alkylene)-, -arylene-, —C₁-C₁₀alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀alkylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈heterocyclo-, —C₁-C₁₀ alkylene-(heterocyclo)-, -(heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—; and r is an integerranging from 1-10, wherein said alkylene groups are unsubstituted andthe remainder of the groups are optionally substituted.

An illustrative stretcher unit is that of Formula IIIa wherein R¹⁷ is—(CH₂)₅— as depicted below (see also U.S. Pat. No. 8,309,093).

Another illustrative stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2, as depicted below (see also U.S.Pat. No. 8,309,093, incorporated by reference herein).

Another illustrative stretcher unit is that of Formula IIIa wherein R¹⁷is arylene- or arylene-C₁-C₁₀ alkylene-. In some embodiments, the arylgroup is an unsubstituted phenyl group. Still, another illustrativestretcher unit is that of Formula IIIb wherein R¹⁷ is —(CH₂)₅—, asdepicted below (see also U.S. Pat. No. 8,309,093, incorporated byreference herein).

In certain embodiments, the stretcher unit is linked to the anti-EGFRantibody via a disulfide bond between a sulfur atom of the anti-EGFRantibody unit and a sulfur atom of the stretcher unit. A representativestretcher unit of this embodiment is depicted within the square bracketsof Formula IV (See below, and see also U.S. Pat. No. 8,309,093,incorporated by reference herein), wherein R¹⁷, L-, —W—, —Y—, -D, w, andy are as defined above.

It should be noted that the S moiety in the formula shown below (seealso U.S. Pat. No. 8,309,093, incorporated by reference herein) refersto a sulfur atom of the antibody, unless otherwise indicated by thecontext.

In yet other embodiments, the stretcher contains a reactive site thatcan form a bond with a primary or secondary amino group of an antibody.Examples of these reactive sites include but are not limited to,activated esters such as succinimide esters, 4 nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative stretcher units of this embodiment are depicted withinthe square brackets of Formulas Va and Vb (See below (see also U.S. Pat.No. 8,309,093, incorporated by reference herein), wherein R¹⁷, L-, —W—,—Y—, -D, w, and y are as defined above.

In some embodiments, the stretcher contains a reactive site that isreactive to a modified carbohydrate's (—CHO) group that can be presenton an antibody. For example, a carbohydrate can be mildly oxidized usinga reagent such as sodium periodate and the resulting (—CHO) unit of theoxidized carbohydrate can be condensed with a Stretcher that contains afunctionality such as a hydrazide, an oxime, a primary or secondaryamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and anarylhydrazide such as those described by Kaneko et al., 1991,Bioconjugate Chem. 2:133-41. Representative Stretcher units of thisembodiment are depicted within the square brackets of Formulas VIa, VIb,and VIc (See below (see also U.S. Pat. No. 8,309,093, incorporated byreference herein), wherein —R¹⁷—, L-, —W—, —Y—, -D, w and y are asdefined as above.

In some embodiments, a linker component comprises an “amino acid unit”(W). In some such embodiments, the amino acid unit allows for cleavageof the linker by a protease, thereby facilitating release of the drugfrom the immunoconjugate upon exposure to intracellular proteases, suchas lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol.21:778-784). Exemplary amino acid units include, but are not limited to,dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplarydipeptides include, but are not limited to, valine-citrulline (vc orval-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine(fk or phe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In one embodiment, the W amino acid unit is valine-citrulline (vc orval-cit). In another aspect, the amino acid unit is phenylalanine-lysine(i.e., fk). In yet another aspect of the amino acid unit, the amino acidunit is N-methylvaline-citrulline. In yet another aspect, the amino acidunit is 5-aminovaleric acid, homo phenylalanine lysine,tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine,isonepecotic acid lysine, beta-alanine lysine, glycine serine valineglutamine and isonepecotic acid.

Alternatively, in some embodiments, —W— is a glucuronide unit that linksa stretcher unit to a spacer unit if the stretcher and spacer units arepresent, links a stretcher unit to the drug moiety if the spacer unit isabsent, and links the linker unit to the drug if the stretcher andspacer units are absent. The glucuronide unit includes a site that canbe cleaved by a β-glucuronidase enzyme (See also US 2012/0107332,incorporated by reference herein). In some embodiments, the glucuronideunit comprises a sugar moiety (Su) linked via a glycoside bond (—O′—) toa self-immolative group (Z) of the formula as depicted below (See alsoUS 2012/0107332, incorporated by reference herein).

●Su-O′—Z

The glycosidic bond (—O′—) is typically a β-glucuronidase-cleavage site,such as a bond cleavable by human, lysosomal β-glucuronidase. In thecontext of a glucuronide unit, the term “self-immolative group” refersto a di- or tri-functional chemical moiety that is capable of covalentlylinking together two or three spaced chemical moieties (i.e., the sugarmoiety (via a glycosidic bond), a drug moiety (directly or indirectlyvia a spacer unit), and, in some embodiments, a linker (directly orindirectly via a stretcher unit) into a stable molecule. Theself-immolative group will spontaneously separate from the firstchemical moiety (e.g., the spacer or drug unit) if its bond to the sugarmoiety is cleaved.

In some embodiments, the sugar moiety (Su) is cyclic hexose, such as apyranose, or a cyclic pentose, such as a furanose. In some embodiments,the pyranose is a glucuronide or hexose. The sugar moiety is usually inthe β-D conformation. In a specific embodiment, the pyranose is aβ-D-glucuronide moiety (i.e., β-D-glucuronic acid linked to theself-immolative group —Z— via a glycosidic bond that is cleavable byβ-glucuronidase). In some embodiments, the sugar moiety is unsubstituted(e.g., a naturally occurring cyclic hexose or cyclic pentose). In otherembodiments, the sugar moiety can be a substituted β-D-glucuronide(i.e., glucuronic acid substituted with one or more group, suchhydrogen, hydroxyl, halogen, sulfur, nitrogen or lower alkyl.

In some embodiments, the glucuronide unit has one of the formulas asdepicted below (See also US 2012/0107332, incorporated by referenceherein),

wherein Su is the sugar moiety, the glycosidic bond comprises the oxygenbond between Su and the self immolative group Z, and each R isindependently hydrogen, halo (e.g., chloro, bromo, fluoro, etc), —CN,—NO₂, or other electron withdrawing or donating group, provided that theGlucuronide unit (and Z in particular) undergoes self-immolation uponcleavage of the glycosidic bond. In some embodiments, each R isindependently hydrogen, halo (e.g., chloro, bromo, fluoro, etc), —CN or—NO₂.

In some embodiments, the glucuronide unit has one of the formulas asdepicted below (see also US 2012/0107332, incorporated by referenceherein),

wherein Su is the Sugar moiety, the glycosidic bond (—O′—) comprises theoxygen bond between Su and the self immolative group Z, and each R isindependently hydrogen.

In some embodiments, the self-immolative group (Z) is covalently linkedto the sugar moiety, to the drug (directly or indirectly via the spacerunit(s)), and to the linker (directly or indirectly via the stretcherunit(s)). In some embodiments, a drug linker conjugate has the formulaas depicted below (See also US 2012/0107332, incorporated by referenceherein),

wherein Su, O′, Z, Y, y, D, A and a are defined herein. Typically from 1to 20 of such drug-linker conjugates can be linked to a linker.

In some embodiments, an ADC comprising the glucuronide unit has one ofthe formulas as depicted below (See also US 2012/0107332, incorporatedby reference herein), wherein Su, Y, y, D, A, a, and L are defined asdescribed herein.

In some embodiments, an ADC comprising the glucuronide unit has theformula as depicted below (See also US 2012/0107332, incorporated byreference herein), wherein Y, y, D, A, a, and L are defined herein.

In some embodiments, an ADC comprising the Glucuronide unit has theformula as depicted below (See also US 2012/0107332, incorporated byreference herein), wherein Y, y, D and L are defined as describedherein.

In some embodiments, an ADC comprising the Glucuronide unit has theformula as depicted below (See also US 2012/0107332, incorporated byreference herein), wherein Y, y, D and L are defined as describedherein.

In some embodiments, an ADC comprising the Glucuronide unit has theformula as depicted below (See also US 2012/0107332 A1), wherein D is asdescribed herein and mAb is a monoclonal antibody.

The spacer unit (—Y—), when present, links an amino acid unit (orGlucuronide unit, see also US 2012/0107332, incorporated by referenceherein) to the drug moiety when an amino acid unit is present.Alternately, the spacer unit links the stretcher unit to the drug moietywhen the amino acid unit is absent. The spacer unit may also links thedrug unit to the antibody unit when both the amino acid unit andstretcher unit are absent.

Spacer units are of two general types: non self-immolative orself-immolative. A non self-immolative spacer unit is one in which partor all of the spacer unit remains bound to the drug moiety aftercleavage, particularly enzymatic, of an amino acid unit (or glucuronideunit) from the antibody-drug conjugate. Examples of a nonself-immolative spacer unit include, but are not limited to a(glycine-glycine) spacer unit and a glycine spacer unit (both depictedin Scheme 1 below (see also U.S. Pat. No. 8,309,093, incorporated byreference herein)).

When a conjugate containing a glycine-glycine spacer unit or a glycinespacer unit undergoes enzymatic cleavage via an enzyme (e.g., atumor-cell associated-protease, a cancer-cell-associated protease or alymphocyte-associated protease), a glycine-glycine-drug moiety or aglycine-drug moiety is cleaved from L-A_(a)-W_(W)—. In one embodiment,an independent hydrolysis reaction takes place within the target cell,cleaving the glycine-drug moiety bond and liberating the drug.

In some embodiments, a non self-immolative spacer unit (—Y—) is -Gly-.In some embodiments, a non self-immolative spacer unit (—Y—) is-Gly-Gly-.

In one embodiment, a drug-linker conjugate is provided in which thespacer unit is absent (y=0), or a pharmaceutically acceptable salt orsolvate thereof.

Alternatively, a conjugate containing a self-immolative spacer unit canallow for the release of the drug moiety. A self-immolative spacer unitwill spontaneously separate from the second chemical moiety if its bondto the first moiety is cleaved.

In some embodiments, —Y_(y)— is a p-aminobenzyl alcohol (PAB) unit whosephenylene portion is substituted with Q_(m), wherein Q is —C₁-C₈ alkyl,—C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl),—O—(C₁-C₈ alkynyl), -halogen, -nitro or -cyano; and m is an integerranging from 0-4. The alkyl, alkenyl and alkynyl groups, whether aloneor as part of another group, can be optionally substituted.

In some embodiments, —Y— is a PAB group that is linked to —W_(w)— viathe amino nitrogen atom of the PAB group, and connected directly to -Dvia a carbonate, carbamate or ether group. Without being bound by anyparticular theory or mechanism, Scheme 2 below (see also U.S. Pat. No.8,309,093) depicts a possible mechanism of drug release of a PAB groupwhich is attached directly to -D via a carbamate or carbonate group asdescribed by Toki et al., 2002, J. Org. Chem. 67:1866-1872.

In Scheme 2, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1to about 20. The alkyl, alkenyl and alkynyl groups, whether alone or aspart of another group, can be optionally substituted.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al., 1999,Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues et al., 1995, Chemistry Biology 2:223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm etal., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acidamides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the α-position of glycine(Kingsbury et al., 1984, J. Med. Chem. 27:1447) are also examples ofself-immolative spacers.

In one aspect, spacer units (—Y_(y)—) are represented by Formulas(X)-(XII) (See below (see also U.S. Pat. No. 8,309,093) wherein Q is—C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈ alkyl),—O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen, -nitro or -cyano; andm is an integer ranging from 0-4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (see, e.g., Hay et al.,1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (see, e.g., Rodrigues et al., 1995, ChemistryBiology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (see, e.g., Storm et al., 1972, J. Amer.Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (see, e.g.,Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the a-position of glycine(see, e.g., Kingsbury et al., 1984, J. Med. Chem. 27:1447) are alsoexamples of self-immolative spacers.

Other suitable spacer units are disclosed in Published U.S. PatentApplication No. 2005-0238649, the disclosure of which is incorporated byreference herein.

Another approach for the generation of ADCs involves the use ofheterobifunctional cross-linkers which link the anti-EGFR antibody tothe drug moiety. Examples of cross-linkers that may be used includeN-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate or the highlywater-soluble analog N-sulfosuccinimidyl4-(5-nitro-2-pyridyldithio)-pentanoate,N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB),N-succinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SNPB), andN-sulfosuccinimidyl-4-(5-nitro-2-pyridyldithio) butyrate (SSNPB),N-succinimidyl-4-methyl-4-(5-nitro-2-pyridyldithio)pentanoate (SMNP),N-succinimidyl-4-(5-N,N-dimethylcarboxamido-2-pyridyldithio) butyrate(SCPB) orN-sulfosuccinimidyl4-(5-N,N-dimethylcarboxamido-2-pyridyldithio)butyrate (SSCPB)). The antibodies of the invention may be modified withthe cross-linkers N-succinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate,N-sulfosuccinimidyl 4-(5-nitro-2-pyridyldithio)-pentanoate, SPDB, SNPB,SSNPB, SMNP, SCPB, or SSCPB can then react with a small excess of aparticular drug that contains a thiol moiety to give excellent yields ofan ADC. Preferably, the cross-linkers are compounds of the formula asdepicted below (see also U.S. Pat. No. 6,913,748, incorporated byreference herein),

wherein R, R₁, R₂ and R₃ are the same or different and are H, methyl,ethyl, or linear, branched, or cyclic alkyl having 3 to 6 carbon atoms,n is 0 or an integer from 1 to 4, X and Y are the same or different andare H, CONR₄R₅ or NO₂, provided that X and Y are not both H at the sametime, R₄ and R₅ are the same or different and are each H, methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl or tert-butyl, and Zis SO₃ ⁻M⁺ or H, wherein M⁺ represents a metal ion or a tetra alkylammonium ion, provided that when X and/or Y is NO₂, Z is not H.Additional heterobifunctional crosslinkers and methods of making ADCsusing the same are described in U.S. Pat. No. 6,913,748, which isexpressly incorporated by reference herein.

In one embodiment, charged linkers (also referred to as pro-chargedlinkers) are used to conjugate anti-EGFR antibodies to drugs to formADCs. Charged linkers include linkers that become charged after cellprocessing. The presence of a charged group(s) in the linker of aparticular ADC or on the drug after cellular processing provides severaladvantages, such as (i) greater water solubility of the ADC, (ii)ability to operate at a higher concentration in aqueous solutions, (iii)ability to link a greater number of drug molecules per antibody,potentially resulting in higher potency, (iv) potential for the chargedconjugate species to be retained inside the target cell, resulting inhigher potency, and (v) improved sensitivity of multidrug resistantcells, which would be unable to export the charged drug species from thecell. Examples of some suitable charged or pro-charged cross-linkers andtheir synthesis are shown in FIGS. 1 to 10 of U.S. Pat. No. 8,236,319,and are incorporated by reference herein. Preferably, the charged orpro-charged cross-linkers are those containing sulfonate, phosphate,carboxyl or quaternary amine substituents that significantly increasethe solubility of the ADCs, especially for ADCs with 2 to 20 conjugateddrugs. Conjugates prepared from linkers containing a pro-charged moietywould produce one or more charged moieties after the conjugate ismetabolized in a cell.

In a further embodiment, the ADC of the invention comprises a linkerhaving the formula as depicted below (see also U.S. Pat. No. 8,236,319,incorporated by reference herein),

wherein Y′ represents a functional group that enables reaction with anantibody; Q represents a functional group that enables linkage of a drugvia a disulfide, thioether, thioester, peptide, hydrazone, ester, ether,carbamate or amide bond; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, and R₁₀ arethe same or different and are H, linear alkyl having from 1 to 6 carbonatoms, branched or cyclic alkyl having from 3 to 6 carbon atoms, linear,branched or cyclic alkenyl or alkynyl having from 2 to 6 carbon atoms,anions, such as but not limited to, SO₃ ⁻, X—SO₃ ⁻, OPO₃ ²⁻, X—OPO₃ ²⁻,PO₃ ²⁻, X—PO₃ ²⁻, CO₂—, cations, such as but not limited to, a nitrogencontaining heterocycle, N⁺R₁₁R₁₂R₁₃, or X—N⁺R₁₁R₁₂R₁₃ or a phenyl,wherein: R₁₁, R₁₂, and R₁₃ are the same or different and are H, linearalkyl having from 1 to 6 carbon atoms, or branched or cyclic alkylhaving from 3 to 6 carbon atoms and X represents phenyl or a linearalkyl having from 1 to 6 carbon atoms, or a branched or cyclic alkylhaving from 3 to 6 carbon atoms; 1, m, and n are 0 or an integer from 1to 4; A is a phenyl or substituted phenyl, wherein the substituent is alinear alkyl having from 1 to 6 carbon atoms, or a branched or cyclicalkyl having from 3 to 6 carbon atoms, or a charged substituent selectedfrom anions, such as but not limited to, SO₃ ⁻, X—SO₃ ⁻, OPO₃ ²⁻, X—OPO₃²⁻, PO₃ ²⁻, X—PO₃ ²⁻, CO₂—, and cations, such as but not limited to, anitrogen containing heterocycle, N⁺R₁₁R₁₂R₁₃ or X—N⁺R₁₁R₁₂R₁₃, wherein Xhas the same definition as above, and wherein g is 0 or 1; Z is anoptional polyethyleneoxy unit of formula (OCH₂CH₂)_(p), wherein p is 0or an integer from 2 to about 1000, or F1-E1-P-E2-F2 unit in which E1and E2 are the same or different and are C═O, O, or NR14, wherein R₁₄ isH, a linear alkyl having from 1 to 6 carbon atoms, a branched or cyclicalkyl having from 3 to 6 carbon atoms, a linear, branched or cyclicalkenyl or alkynyl having from 2 to 6 carbon atoms; P is a peptide unitbetween 2 and 20 amino acids in length, wherein E1 or E2 can be linkedto the peptide through the terminal nitrogen, terminal carbon or througha side chain of one of the amino acids of the peptide; and F1 and F2 arethe same or different and are an optional polyethyleneoxy unit offormula (OCH₂CH₂)_(p), wherein p is 0 or an integer from 2 to about1000, provided that when Z is not F1-E1-P-E2-F2, at least one of R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, and R₁₀ is a charged substituent or when gis 1, at least one of A, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, and R₁₀ isa charged substituent.

Additional examples of linkers that can be used with the compositionsand methods include valine-citrulline; maleimidocaproyl; amino benzoicacids; p-aminobenzylcarbamoyl (PAB); lysosomal enzyme-cleavable linkers;maleimidocaproyl-polyethylene glycol (MC(PEG)6-OH); N-methyl-valinecitrulline; N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); N-Succinimidyl4-(2-pyridyldithio)butanoate (SPDB); and N-Succinimidyl4-(2-pyridylthio)pentanoate (SPP) (See also US 2011/0076232). Anotherlinker for use in the invention includes an avidin-biotin linkage toprovide an avidin-biotin-containing ADC (See also U.S. Pat. No.4,676,980, PCT publication Nos. WO1992/022332A2, WO1994/016729A1,WO1995/015770A1, WO1997/031655A2, WO1998/035704A1, WO1999/019500A1,WO2001/09785A2, WO2001/090198A1, WO2003/093793A2, WO2004/050016A2,WO2005/081898A2, WO2006/083562A2, WO2006/089668A1, WO2007/150020A1,WO2008/135237A1, WO2010/111198A1, WO2011/057216A1, WO2011/058321A1,WO2012/027494A1, and EP77671B1), wherein some such linkers are resistantto biotinidase cleavage. Additional linkers that may be used in theinvention include a cohesin/dockerin pair to provide acohesion-dockerin-containing ADC (See PCT publication Nos.WO2008/097866A2, WO2008/097870A2, WO2008/103947A2, and WO2008/103953A2).

Additional linkers for use in the invention may contain non-peptidepolymers (examples include, but are not limited to, polyethylene glycol,polypropylene glycol, polyoxyethylated polyols, polyvinyl alcohol,polysaccharides, dextran, polyvinyl ethyl ether, PLA (poly(lacticacid)), PLGA (poly(lactic acid-glycolic acid)), and combinationsthereof, wherein a preferred polymer is polyethylene glycol) (See alsoPCT publication No. WO2011/000370). Additional linkers are alsodescribed in WO 2004-010957, U.S. Publication No. 20060074008, U.S.Publication No. 20050238649, and U.S. Publication No. 20060024317, eachof which is incorporated by reference herein in its entirety).

For an ADC comprising a maytansinoid, many positions on maytansinoidscan serve as the position to chemically link the linking moiety. In oneembodiment, maytansinoids comprise a linking moiety that contains areactive chemical group are C-3 esters of maytansinol and its analogswhere the linking moiety contains a disulfide bond and the chemicalreactive group comprises a N-succinimidyl or N-sulfosuccinimidyl ester.For example, the C-3 position having a hydroxyl group, the C-14 positionmodified with hydroxymethyl, the C-15 position modified with hydroxy andthe C-20 position having a hydroxy group are all useful. The linkingmoiety most preferably is linked to the C-3 position of maytansinol.

The conjugation of the drug to the antibody via a linker can beaccomplished by any technique known in the art. A number of differentreactions are available for covalent attachment of drugs and linkers toantibodies. This may be accomplished by reaction of the amino acidresidues of the antibody, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.One of the most commonly used non-specific methods of covalentattachment is the carbodiimide reaction to link a carboxy (or amino)group of a compound to amino (or carboxy) groups of the antibody.Additionally, bifunctional agents such as dialdehydes or imidoestershave been used to link the amino group of a compound to amino groups ofan antibody. Also available for attachment of drugs to antibodies is theSchiff base reaction. This method involves the periodate oxidation of adrug that contains glycol or hydroxy groups, thus forming an aldehydewhich is then reacted with the binding agent. Attachment occurs viaformation of a Schiff base with amino groups of the antibody.Isothiocyanates can also be used as coupling agents for covalentlyattaching drugs to antibodies. Other techniques are known to the skilledartisan and within the scope of the invention.

In certain embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with theanti-EGFR antibody under appropriate conditions. The synthesis andstructure of exemplary linkers, stretcher units, amino acid units,self-immolative spacer units are described in U.S. Patent ApplicationPublication Nos. 20030083263, 20050238649 and 20050009751, each if whichis incorporated herein by reference.

Stability of the ADC may be measured by standard analytical techniquessuch as mass spectroscopy, HPLC, and the separation/analysis techniqueLC/MS.

IV. Purification of Anti-EGFR ADCs

Purification of the ADCs may be achieved in such a way that ADCs havingcertain DARs are collected. For example, HIC resin may be used toseparate high drug loaded ADCs from ADCs having optimal drug to antibodyratios (DARs), e.g. a DAR of 4 or less. In one embodiment, a hydrophobicresin is added to an ADC mixture such that undesired ADCs, i.e., higherdrug loaded ADCs, bind the resin and can be selectively removed from themixture. In certain embodiments, separation of the ADCs may be achievedby contacting an ADC mixture (e.g., a mixture comprising a drug loadedspecies of ADC of 4 or less and a drug loaded species of ADC of 6 ormore) with a hydrophobic resin, wherein the amount of resin issufficient to allow binding of the drug loaded species which is beingremoved from the ADC mixture. The resin and ADC mixture are mixedtogether, such that the ADC species being removed (e.g., a drug loadedspecies of 6 or more) binds to the resin and can be separated from theother ADC species in the ADC mixture. The amount of resin used in themethod is based on a weight ratio between the species to be removed andthe resin, where the amount of resin used does not allow for significantbinding of the drug loaded species that is desired. Thus, methods may beused to reduce the average DAR 5.5 to less than 4. Further, thepurification methods described herein may be used to isolate ADCs havingany desired range of drug loaded species, e.g., a drug loaded species of4 or less, a drug loaded species of 3 or less, a drug loaded species of2 or less, a drug loaded species of 1 or less.

Certain species of molecule(s) binds to a surface based on hydrophobicinteractions between the species and a hydrophobic resin. In oneembodiment, method of the invention refers to a purification processthat relies upon the intermixing of a hydrophobic resin and a mixture ofADCs, wherein the amount of resin added to the mixture determines whichspecies (e.g., ADCs with a DAR of 6 or more) will bind. Followingproduction and purification of an antibody from an expression system(e.g., a mammalian expression system), the antibody is reduced andcoupled to a drug through a conjugation reaction. The resulting ADCmixture often contains ADCs having a range of DARs, e.g., 1 to 8. In oneembodiment, the ADC mixture comprises a drug loaded species of 4 or lessand a drug loaded species of 6 or more. According to the methods of theinvention, the ADC mixture may be purified using a process, such as, butnot limited to, a batch process, such that ADCs having a drug loadedspecies of 4 or less are selected and separated from ADCs having ahigher drug load (e.g., ADCs having a drug loaded species of 6 or more).Notably, the purification methods described herein may be used toisolate ADCs having any desired range of DAR, e.g., a DAR of 4 or less,a DAR of 3 or less, a DAR of 2 or less.

Thus, in one embodiment, an ADC mixture comprising a drug loaded speciesof 4 or less and a drug loaded species of 6 or more may be contactedwith a hydrophobic resin to form a resin mixture, wherein the amount ofhydrophobic resin contacted with the ADC mixture is sufficient to allowbinding of the drug loaded species of 6 or more to the resin but doesnot allow significant binding of the drug load species of 4 or less; andremoving the hydrophobic resin from the ADC mixture, such that thecomposition comprising ADCs is obtained, wherein the compositioncomprises less than 15% of the drug loaded species of 6 or more, andwherein the ADC comprises an antibody conjugated to an auristatin. In aseparate embodiment, the method of the invention comprises contacting anADC mixture comprising a drug loaded species of 4 or less and a drugloaded species of 6 or more with a hydrophobic resin to form a resinmixture, wherein the amount of hydrophobic resin contacted with the ADCmixture is sufficient to allow binding of the drug loaded species of 6or more to the resin but does not allow significant binding of the drugload species of 4 or less; and removing the hydrophobic resin from theADC mixture, such that the composition comprising ADCs is obtained,wherein the composition comprises less than 15% of the drug loadedspecies of 6 or more, and wherein the ADC comprises an antibodyconjugated to an auristatin, wherein the hydrophobic resin weight is 3to 12 times the weight of the drug loaded species of 6 or more in theADC mixture.

The ADC separation method described herein method may be performed usinga batch purification method. The batch purification process generallyincludes adding the ADC mixture to the hydrophobic resin in a vessel,mixing, and subsequently separating the resin from the supernatant. Forexample, in the context of batch purification, a hydrophobic resin maybe prepared in or equilibrated to the desired equilibration buffer. Aslurry of the hydrophobic resin may thus be obtained. The ADC mixturemay then be contacted with the slurry to adsorb the specific species ofADC(s) to be separated by the hydrophobic resin. The solution comprisingthe desired ADCs that do not bind to the hydrophobic resin material maythen be separated from the slurry, e.g., by filtration or by allowingthe slurry to settle and removing the supernatant. The resulting slurrycan be subjected to one or more washing steps. In order to elute boundADCs, the salt concentration can be decreased. In one embodiment, theprocess used in the invention includes no more than 50 g of hydrophobicresin.

Thus, a batch method may be used to contact an ADC mixture comprising adrug loaded species of 4 or less and a drug loaded species of 6 or morewith a hydrophobic resin to form a resin mixture, wherein the amount ofhydrophobic resin contacted with the ADC mixture is sufficient to allowbinding of the drug loaded species of 6 or more to the resin but doesnot allow significant binding of the drug load species of 4 or less; andremoving the hydrophobic resin from the ADC mixture, such that thecomposition comprising ADCs is obtained, wherein the compositioncomprises less than 15% of the drug loaded species of 6 or more, andwherein the ADC comprises an antibody conjugated to an auristatin. In aseparate embodiment, a batch method is used to contact an ADC mixturecomprising a drug loaded species of 4 or less and a drug loaded speciesof 6 or more with a hydrophobic resin to form a resin mixture, whereinthe amount of hydrophobic resin contacted with the ADC mixture issufficient to allow binding of the drug loaded species of 6 or more tothe resin but does not allow significant binding of the drug loadspecies of 4 or less; and removing the hydrophobic resin from the ADCmixture, such that the composition comprising ADCs is obtained, whereinthe composition comprises less than 15% of the drug loaded species of 6or more, and wherein the ADC comprises an antibody conjugated to anauristatin, wherein the hydrophobic resin weight is 3 to 12 times theweight of the drug loaded species of 6 or more in the ADC mixture.

Alternatively, in a separate embodiment, purification may be performedusing a circulation process, whereby the resin is packed in a containerand the ADC mixture is passed over the hydrophobic resin bed until thespecific species of ADC(s) to be separated have been removed. Thesupernatant (containing the desired ADC species) is then pumped from thecontainer and the resin bed may be subjected to washing steps.

A circulation process may be used to contact an ADC mixture comprising adrug loaded species of 4 or less and a drug loaded species of 6 or morewith a hydrophobic resin to form a resin mixture, wherein the amount ofhydrophobic resin contacted with the ADC mixture is sufficient to allowbinding of the drug loaded species of 6 or more to the resin but doesnot allow significant binding of the drug load species of 4 or less; andremoving the hydrophobic resin from the ADC mixture, such that thecomposition comprising ADCs is obtained, wherein the compositioncomprises less than 15% of the drug loaded species of 6 or more, andwherein the ADC comprises an antibody conjugated to an auristatin. In aseparate embodiment, a circulation process is used to contact an ADCmixture comprising a drug loaded species of 4 or less and a drug loadedspecies of 6 or more with a hydrophobic resin to form a resin mixture,wherein the amount of hydrophobic resin contacted with the ADC mixtureis sufficient to allow binding of the drug loaded species of 6 or moreto the resin but does not allow significant binding of the drug loadspecies of 4 or less; and removing the hydrophobic resin from the ADCmixture, such that the composition comprising ADCs is obtained, whereinthe composition comprises less than 15% of the drug loaded species of 6or more, and wherein the ADC comprises an antibody conjugated to anauristatin, wherein the hydrophobic resin weight is 3 to 12 times theweight of the drug loaded species of 6 or more in the ADC mixture.

Alternatively, a flow through process may be used to purify an ADCmixture to arrive at a composition comprising a majority of ADCs havinga certain desired DAR. In a flow through process, resin is packed in acontainer, e.g., a column, and the ADC mixture is passed over the packedresin such that the desired ADC species does not substantially bind tothe resin and flows through the resin, and the undesired ADC species isbound to the resin. A flow through process may be performed in a singlepass mode (where the ADC species of interest are obtained as a result ofa single pass through the resin of the container) or in a multi-passmode (where the ADC species of interest are obtained as a result ofmultiple passes through the resin of the container). The flow throughprocess is performed such that the weight of resin selected binds to theundesired ADC population, and the desired ADCs (e.g., DAR 2-4) flow overthe resin and are collected in the flow through after one or multiplepasses.

A flow through process may be used to contact an ADC mixture comprisinga drug loaded species of 4 or less and a drug loaded species of 6 ormore with a hydrophobic resin, wherein the amount of hydrophobic resincontacted with the ADC mixture is sufficient to allow binding of thedrug loaded species of 6 or more to the resin but does not allowsignificant binding of the drug load species of 4 or less, where thedrug load species of 4 or less passes over the resin and is subsequentlycollected after one or multiple passes, such that the compositioncomprising the desired ADCs (e.g. DAR 2-4) is obtained, wherein thecomposition comprises less than 15% of the drug loaded species of 6 ormore, and wherein the ADC comprises an antibody conjugated to anauristatin. In a separate embodiment, a flow through process is used tocontact an ADC mixture comprising a drug loaded species of 4 or less anda drug loaded species of 6 or more with a hydrophobic resin by passingthe ADC mixture over the resin, wherein the amount of hydrophobic resincontacted with the ADC mixture is sufficient to allow binding of thedrug loaded species of 6 or more to the resin but does not allowsignificant binding of the drug load species of 4 or less, where thedrug load species of 4 or less passes over the resin and is subsequentlycollected, such that the composition comprising ADCs is obtained,wherein the composition comprises less than 15% of the drug loadedspecies of 6 or more, and wherein the ADC comprises an antibodyconjugated to an auristatin, wherein the amount of hydrophobic resinweight is 3 to 12 times the weight of the drug loaded species of 6 ormore in the ADC mixture.

Following a flow through process, the resin may be washed with a one ormore washes following in order to further recover ADCs having thedesired DAR range (found in the wash filtrate). For example, a pluralityof washes having decreasing conductivity may be used to further recoverADCs having the DAR of interest. The elution material obtained from thewashing of the resin may be subsequently combined with the filtrateresulting from the flow through process for improved recovery of ADCshaving the DAR of interest.

The aforementioned batch, circulation, and flow through processpurification methods are based on the use of a hydrophobic resin toseparate high vs. low drug loaded species of ADC. Hydrophobic resincomprises hydrophobic groups which interact with the hydrophobicproperties of the ADCs. Hydrophobic groups on the ADC interact withhydrophobic groups within the hydrophobic resin. The more hydrophobic aprotein is the stronger it will interact with the hydrophobic resin.

Hydrophobic resin normally comprises a base matrix (e.g., cross-linkedagarose or synthetic copolymer material) to which hydrophobic ligands(e.g., alkyl or aryl groups) are coupled. Many hydrophobic resins areavailable commercially. Examples include, but are not limited to, PhenylSepharose™ 6 Fast Flow with low or high substitution (Pharmacia LKBBiotechnology, AB, Sweden); Phenyl Sepharose™ High Performance(Pharmacia LKB Biotechnology, AB, Sweden); Octyl Sepharose™ HighPerformance (Pharmacia LKB Biotechnology, AB, Sweden); Fractogel™ EMDPropyl or Fractogel™ EMD Phenyl columns (E. Merck, Germany); Macro-Prep™Methyl or Macro-Prep™. t-Butyl Supports (Bio-Rad, California); WPHI-Propyl (C₃)™ (J. T. Baker, New Jersey); and Toyopearl™ ether, hexyl,phenyl or butyl (TosoHaas, PA). In one embodiment, the hydrophobic resinis a butyl hydrophobic resin. In another embodiment, the hydrophobicresin is a phenyl hydrophobic resin. In another embodiment, thehydrophobic resin is a hexyl hydrophobic resin, an octyl hydrophobicresin, or a decyl hydrophobic resin. In one embodiment, the hydrophobicresin is a methacrylic polymer having n-butyl ligands (e.g. TOYOPEARL″Butyl-600M).

Further methods for purifying ADC mixtures to obtain a compositionhaving a desired DAR are described in U.S. application Ser. No.14/210,602 (U.S. Patent Appln. Publication No. US 2014/0286968),incorporated by reference in its entirety.

V. Uses of Anti-EGFR Antibodies and Anti-EGFR ADCs

The antibodies and antibody portions (and ADCs) of the inventionpreferably are capable of neutralizing human EGFR activity both in vivo.Accordingly, such antibodies and antibody portions of the invention canbe used to inhibit hEGFR activity, e.g., in a cell culture containinghEGFR, in human subjects or in other mammalian subjects having EGFR withwhich an antibody of the invention cross-reacts. In one embodiment, theinvention provides a method for inhibiting hEGFR activity comprisingcontacting hEGFR with an antibody or antibody portion of the inventionsuch that hEGFR activity is inhibited. For example, in a cell culturecontaining, or suspected of containing hEGFR, an antibody or antibodyportion of the invention can be added to the culture medium to inhibithEGFR activity in the culture.

In another embodiment, of the invention a method for reducing hEGFRactivity in a subject, advantageously from a subject suffering from adisease or disorder in which EGFR activity is detrimental. The inventionprovides methods for reducing EGFR activity in a subject suffering fromsuch a disease or disorder, which method comprises administering to thesubject an antibody or antibody portion of the invention such that EGFRactivity in the subject is reduced. Preferably, the EGFR is human EGFR,and the subject is a human subject. Alternatively, the subject can be amammal expressing a EGFR to which antibodies of the invention arecapable of binding. Still further the subject can be a mammal into whichEGFR has been introduced (e.g., by administration of EGFR or byexpression of a EGFR transgene). Antibodies of the invention can beadministered to a human subject for therapeutic purposes. Moreover,antibodies of the invention can be administered to a non-human mammalexpressing a EGFR with which the antibody is capable of binding forveterinary purposes or as an animal model of human disease. Regardingthe latter, such animal models may be useful for evaluating thetherapeutic efficacy of antibodies of the invention (e.g., testing ofdosages and time courses of administration).

As used herein, the term “a disorder in which EGFR activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of EGFR in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which EGFRactivity is detrimental is a disorder in which reduction of EGFRactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of EGFR in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofEGFR in a tumor, serum, plasma, synovial fluid, etc. of the subject),which can be detected, for example, using an anti-EGFR antibody asdescribed above. Non-limiting examples of disorders that can be treatedwith the antibodies of the invention, for example, AbA, or antigenbinding fragments thereof, include those disorders discussed below. Forexample, suitable disorders include, but are not limited to, a varietyof cancers including, but not limited to, breast cancer, lung cancer, aglioma, prostate cancer, pancreatic cancer, colon cancer, head and neckcancer, and kidney cancer. Other examples of cancer that may be treatedusing the compositions and methods disclosed herein include squamouscell carcinoma (e.g., squamous lung cancer or squamous head and neckcancer), triple negative breast cancer, non-small cell lung cancer.colorectal cancer, and mesothelioma. In one embodiment, the antibodiesand ADCs disclosed herein are used to treat a solid tumor, e.g., inhibitgrowth of or decrease size of a solid tumor, overexpressing EGFR orwhich is EGFR positive. In one embodiment, the invention is directed tothe treatment of EGFR amplified squamous lung cancer. In one embodiment,the antibodies and ADCs disclosed herein are used to treat EGFRamplified squamous head and neck cancer. In another embodiment, theantibodies and ADCs disclosed herein are used to treat triple negativebreast cancer (TNBC). Diseases and disorders described herein may betreated by anti-EGFR antibodies or ADCs of the invention, as well aspharmaceutical compositions comprising such anti-EGFR antibodies orADCs.

In certain embodiments, the antibodies and ADCs disclosed herein areadministered to a subject in need thereof in order to treat advancedsolid tumor types likely to exhibit elevated levels of Epidermal GrowthFactor Receptor (EGFR). Examples of such tumors include, but are notlimited to, head and neck squamous cell carcinoma, non-small cell lungcancer, triple negative breast cancer, colorectal carcinoma, andglioblastoma multiforme.

In certain embodiments, the invention includes a method for inhibitingor decreasing solid tumor growth in a subject having a solid tumor, saidmethod comprising administering an anti-EGFR antibody or ADC describedherein, to the subject having the solid tumor, such that the solid tumorgrowth is inhibited or decreased. In certain embodiments, the solidtumor is a non-small cell lung carcinoma or a glioblastoma. In furtherembodiments, the solid tumor is an EGFRvIII positive tumor or anEGFR-expressing solid tumors. In further embodiments, the solid tumor isan EGFR amplified solid tumor or an EGFR overexpressing solid tumors. Incertain embodiments the anti-EGFR antibodies or ADCs described hereinare are administered to a subject having glioblastima multiforme, aloneor in combination with an additional agent, e.g., radiation and/ortemozolomide.

In certain embodiments, the invention includes a method for inhibitingor decreasing solid tumor growth in a subject having a solid tumor whichwas identified as an EGFR expressing or EGFR overexpressing tumor (or anEGFRvIII expressing tumor), said method comprising administering ananti-EGFR antibody or ADC described herein, to the subject having thesolid tumor, such that the solid tumor growth is inhibited or decreased.Methods for identifying EGFR expressing tumors (e.g., EGFRoverexpressing tumors) are known in the art, and include FDA-approvedtests and validation assays. For example, the EGFR pharmDx™ assay (DakoNorth America, Inc.) is a qualitative immunohistochemical (IHC) kitsystem used to identify EGFR expression in normal and neoplastic tissuesroutinely-fixed for histological evaluation. EGFR pharmDx specificallydetects the EGFR (HER1) protein in EGFR-expressing cells. In addition,PCR-based assays may also be used for identifying EGFR overexpressingtumors. For example, these assays may use primers that are specific forthe variant EGFR gene (e.g., SEQ ID NO: 33) and/or cDNA and result inthe amplification of the EGFR gene/cDNA, or a portion thereof. Theamplified PCR products may be subsequently analyzed, for example, by gelelectrophoresis using standard methods known in the art to determine thesize of the PCR products. Such tests may be used to identify tumors thatmay be treated with the methods and compositions described herein.

Any of the methods for gene therapy available in the art can be usedaccording to the invention. For general reviews of the methods of genetherapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wuand Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan, Science 260:926-932 (1993); and Morganand Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH11(5):155-215. Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley &Sons, N Y (1993);and Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990). Detailed description of various methods ofgene therapy is provided in US20050042664 A1 which is incorporatedherein by reference.

In another aspect, this application features a method of treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing a EGFR-associateddisorder, in a subject. The method includes: administering to thesubject an EGFR binding agent (particularly an antagonist), e.g., ananti-EGFR antibody or fragment thereof as described herein, in an amountsufficient to treat or prevent the EGFR-associated disorder. The EGFRantagonist, e.g., the anti-EGFR antibody or fragment thereof, can beadministered to the subject, alone or in combination with othertherapeutic modalities as described herein.

Antibodies or ADCs of the invention, or antigen binding portions thereofcan be used alone or in combination to treat such diseases. It should beunderstood that the antibodies of the invention or antigen bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the invention. The additional agent also can be an agent that impartsa beneficial attribute to the therapeutic composition, e.g., an agentwhich affects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the invention and atleast one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

The combination therapy can include one or more EGFR antagonists, e.g.,anti-EGFR antibodies or fragments thereof, formulated with, and/orco-administered with, one or more additional therapeutic agents, e.g.,one or more cytokine and growth factor inhibitors, immunosuppressants,anti-inflammatory agents (e.g., systemic anti-inflammatory agents),anti-fibrotic agents, metabolic inhibitors, enzyme inhibitors, and/orcytotoxic or cytostatic agents, mitotic inhibitors, antitumorantibiotics, immunomodulating agents, vectors for gene therapy,alkylating agents, antiangiogenic agents, antimetabolites,boron-containing agents, chemoprotective agents, hormones, antihormoneagents, corticosteroids, photoactive therapeutic agents,oligonucleotides, radionuclide agents, topoisomerase inhibitors,tyrosine kinase inhibitors, or radiosensitizers, as described in moreherein.

In a particular embodiment, the anti-EGFR binding proteins describedherein, for example, anti-EGFR antibodies, are used in combination withan anti-cancer agent or an antineoplastic agent. The terms “anti-canceragent” and “antineoplastic agent” refer to drugs used to treatmalignancies, such as cancerous growths. Drug therapy may be used alone,or in combination with other treatments such as surgery or radiationtherapy. Several classes of drugs may be used in cancer treatment,depending on the nature of the organ involved. For example, breastcancers are commonly stimulated by estrogens, and may be treated withdrugs which inactive the sex hormones. Similarly, prostate cancer may betreated with drugs that inactivate androgens, the male sex hormone.Anti-cancer agents that may be used in conjunction with the anti-EGFRantibodies or ADCs of the invention include, among others, the followingagents:

Anti-Cancer Agent Comments Examples Antibodies Antibodies which bindIGF- A12 (fully humanized mAb) (a) antibodies other 1R (insulin-likegrowth 19D12 (fully humanized mAb) than anti-EGFR factor type 1receptor), Cp751-871 (fully humanized mAb) antibodies; and which isexpressed on the H7C10 (humanized mAb) (b) anti-EGFR cell surface ofmost human alphaIR3 (mouse) antibodies which cancers ScFV/FC(mouse/human chimera) bind different EM/164 (mouse) epitopes Antibodieswhich bind Matuzumab (EMD72000) EGFR (epiderman growthErbitux ®/Cetuximab (Imclone) factor receptor); MutationsVectibix ®/Panitumumab (Amgen) affecting EGFR expression mAb 806 oractivity could result in Nimotuxumab (TheraCIM) cancer Antibodies whichbind AVEO (AV299) (AVEO) cMET (Mesechymal AMG102 (Amgen) epithelialtransition factor); 5D5 (OA-5d5) (Genentech) a member of the MET H244G11(Pierre Fabre) family of receptor tyrosine kinases) Anti-ErbB3antibodies Ab #14 (MM 121-14) which bind different Herceptin ®(Trastuzumab; Genentech) epitopes 1B4C3; 2D1D12 (U3 Pharma AG) SmallMolecules Insulin-like growth factor NVP-AEW541-A Targeting IGF1R type 1receptor which is BMS-536,924 (1H-benzoimidazol-2-yl)-1H- expressed onthe cell surface pyridin-2-one) of many human cancers BMS-554,417Cycloligan TAE226 PQ401 Small Molecules EGFR (epidermal growthIressa ®/Gefitinib (AstraZeneca) Targeting EGFR factor receptor);CI-1033 (PD 183805) (Pfizer) Overexpression or mutations Lapatinib(GW-572016) (GlaxoSmithKline) affecting EGFR expressionTykerb ®/Lapatinib Ditosylate (Smith Kline or activity could result inBeecham) cancer Tarceva  ®/Erlotinib HCL (OSI-774) (OSI Pharma) PKI-166(Novartis) PD-158780 EKB-569 Tyrphostin AG 1478(4-(3-Chloroanillino)-6,7- dimethoxyquinazoline) Small Molecules cMET(Mesenchymal PHA665752 Targeting cMET epithelial transition factor); ARQ197 a member of the MET family of receptor tyrosine kinases)Antimetabolites Flourouracil (5-FU) Capecitabine/XELODA ® (HLR Roche)5-Trifluoromethyl-2′-deoxyuridine Methotrexate sodium (Trexall) (Barr)Raltitrexed/Tomudex ® (AstraZeneca) Pemetrexed/Alimta ® (Lilly) TegafurCytosine Arabinoside (Cytarabine, Ara-C)/ Thioguanine ®(GlaxoSmithKline) 5-azacytidine 6-mercaptopurine (Mercaptopurine, 6-MP)Azathioprine/Azasan ® (AAIPHARMA LLC) 6-thioguanine (6-TG)/Purinethol ®(TEVA) Pentostatin/Nipent ® (Hospira Inc.) Fludarabinephosphate/Fludara ® (Bayer Health Care) Cladribine (2-CdA,2-chlorodeoxyadenosine)/ Leustatin ® (Ortho Biotech) Alkylating agentsAn alkylating antineoplastic Ribonucleotide Reductase Inhibitor (RNR)agent is an alkylating agent Cyclophosphamide/Cytoxan (BMS) thatattaches an alkyl group Neosar (TEVA) to DNA. Since cancer cellsIfosfamide/Mitoxana ® (ASTA Medica) generally proliferate Thiotepa(Bedford, Abraxis, Teva) unrestrictively more than do BCNU→1,3-bis(2-chloroethyl)-1-nitosourea healthy cells they are more CCNU→1,-(2-chloroethyl)-3-cyclohexyl-1- sensitive to DNA damage, nitrosourea(methyl CCNU) and alkylating agents are Hexamethylmelamine (Altretamine,HMM)/ used clinically to treat a Hexalen ® (MGI Pharma Inc.) variety oftumors. Busulfan/Myleran (GlaxoSmithKline) Procarbazine HCL/ Matulane(Sigma Tau Pharmaceuticals, Inc.) Dacarbazine (DTIC)Chlorambucil/Leukara ® (SmithKline Beecham) Melphalan/Alkeran ®(GlaxoSmithKline) Cisplatin (Cisplatinum, CDDP)/Platinol (Bristol Myers)Carboplatin/Paraplatin (BMS) Oxaliplatin/Eloxitan ® (Sanofi-Aventis US)Topoisomerase Topoisomerase inhibitors Doxorubicin HCL/Doxil ® (Alza)inhibitors are chemotherapy agents Daunorubicin citrate/Daunoxome ®(Gilead) designed to interfere with Mitoxantrone HCL/Novantrone (EMDSerono) the action of topoisomerase Actinomycin D enzymes (topoisomeraseI Etoposide/Vepesid ® (BMS)/Etopophos ® and II), which are enzymes(Hospira, Bedford, Teva Parenteral, Etc.) that control the changes inTopotecan HCL/Hycamtin ® DNA structure by catalyzing (GlaxoSmithKline)the breaking and rejoining Teniposide (VM-26)/Vumon ® (BMS) of thephosphodiester Irinotecan HCL(CPT-ll)/Camptosar ® backbone of DNAstrands (Pharmacia & Upjohn) during the normal cell cycle. Microtubuletargeting Microtubules are one of the Vincristine/Oncovin ® (Lilly)agents components of the Vinblastine sulfate/Velban ® (discontinued)cytoskeleton. They have (Lilly) diameter of ~24 nm and Vinorelbinetartrate/Navelbine ® (PierreFabre) length varying from several Vindesinesulphate/Eldisine ® (Lilly) micrometers to possibly Pac1itaxel/Taxol ®(BMS) millimeters in axons of Docetaxel/Taxotere ® (Sanofi Aventis US)nerve cells. Microtubules Nanoparticle paclitaxel (ABI-007)/Abraxane ®serve as structural (Abraxis BioScience, Inc.) components within cellsand Ixabepilone/IXEMPRA ™ (BMS) are involved in many cellular processesincluding mitosis, cytokinesis, and vesicular transport. Kinaseinhibitors Tyrosine kinases are Imatinib mesylate/Gleevec (Novartis)enzymes within the cell that Sunitinib malate/Sutent ® (Pfizer) functionto attach phosphate Sorafenib toslate/Nexavar ® (Bayer) groups to theamino acid Nilotinib hydrochloride monohydrate/Tasigna ® tyrosine. Byblocking the (Novartis) ability of protein tyrosine kinases to function,these compounds provide a tool for controlling cancerous cell growth.Protein synthesis Induces cell apoptosis L-asparaginase/Elspar ® (Merck& Co.) inhibitors Immunotherapeutic Induces cancer patients to Alphainterferon agents exhibit immune Angiogenesis Inhibitor/Avastin ®(Genentech) responsiveness IL-2→ Interleukin 2 (Aldesleukin)/Proleukin ® (Chiron) IL-12→ Interleukin 12 Antibody/small molecule Anti-CTLA-4and PR-1 therapies immune checkpoint Yervoy ® (ipilimumab; Bristol-MyersSquibb) modulators Opdivo ® (nivolumab; Bristol-Myers Squibb) Keytrada ®(pembrolizumab; Merck) Hormones Hormone therapies Toremifenecitrate/Fareston ® (GTX, Inc.) associated with menopauseFulvestrant/Faslodex ® (AstraZeneca) and aging seek to increaseRaloxifene HCL/Evista ® (Lilly) the amount of certainAnastrazole/Arimidex ® (AstraZeneca) hormones in your body toLetrozole/Femara ® (Novartis) compensate for age- or Fadrozole (CGS16949A) disease-related hormonal Exemestane/Aromasin ® (Pharmacia &Upjohn) declines. Hormone therapy Leuprolide acetate/Eligard ® (QTL USA)as a cancer treatment either Lupron ® (TAP Pharm) reduces the level ofspecific Goserelin acetate/Zoladex ® (AstraZeneca) hormones or altersthe Triptorelin pamoate/Trelstar ® (Watson Labs) cancer's ability to usethese Buserelin/Suprefact ® (Sanofi Aventis) hormones to grow andNafarelin/Synarel ® (Pfizer) spread. Cetrorelix/Cetrotide ® (EMD Serono)Bicalutamide/Casodex ® (AstraZeneca) Nilutamide/Nilandron ® (AventisPharm.) Megestrol acetate/Megace ® (BMS) Somatostatin Analogs(Octreotide acetate/ Sandostatin ® (Novartis) GlucocorticoidsAnti-inflammatory drugs Predinsolone used to reduce swelling thatDexamethasone/Decadron ® (Wyeth) causes cancer pain. Aromatoseinhibitors Includes imidazoles Ketoconazole mTOR inhibitors the mTORsignaling Sirolimus (Rapamycin)/Rapamune ® (Wyeth) pathway wasoriginally Temsirolimus (CCI-779)/Torisel ® (Wyeth) discovered duringstudies of Deforolimus (AP23573)/(Ariad Pharm.) the immunosuppressiveEverolimus (RAD00I)/Certican ® (Novartis) agent rapamycin. This highlyconserved pathway regulates cell proliferation and metabolism inresponse to environmental factors, linking cell growth factor receptorsignaling via phosphoinositide-3- kinase(PI-3K) to cell growth,proliferation, and angiogenesis.

In addition to the above anti-cancer agents, the anti-EGFR antibodiesand ADCs described herein may be administered in combination with theagents described in section II. Further, the aforementioned anti-canceragents may also be used in the ADCs of the invention.

In particular embodiments, the anti-EGFR antibodies or ADCs can beadministered alone or with another anti-cancer agent which acts inconjunction with or synergistically with the antibody to treat thedisease associated with EGFR activity. Such anti-cancer agents include,for example, agents well known in the art (e.g., cytotoxins,chemotherapeutic agents, small molecules and radiation). Examples ofanti-cancer agents include, but are not limited to, Panorex(Glaxo-Welcome), Rituxan (IDEC/Genentech/Hoffman la Roche), Mylotarg(Wyeth), Campath (Millennium), Zevalin (IDEC and Schering AG), Bexxar(Corixa/GSK), Erbitux (Imclone/BMS), Avastin (Genentech) and Herceptin(Genentech/Hoffman la Roche). Other anti-cancer agents include, but arenot limited to, those disclosed in U.S. Pat. No. 7,598,028 andInternational Publication No. WO2008/100624, the contents of which arehereby incorporated by reference. One or more anti-cancer agents may beadministered either simultaneously or before or after administration ofan antibody or antigen binding portion thereof of the invention.

In particular embodiments of the invention, the anti-EGFR antibodies orADCs described herein can be used in a combination therapy with anapoptotic agent, such as a bcl-xl inhibitor or a Bcl-2 (B-cell lymphoma2) inhibitor (e.g., ABT-199 (venetoclax)) to treat cancer, such asleukemia, in a subject. In one embodiment, the anti-EGFR antibodies orADCs described herein can be used in a combination therapy with a bcl-xlinhibitor for treating cancer. In one embodiment, the anti-EGFRantibodies or ADCs described herein can be used in a combination therapywith venetoclax for treating cancer.

In particular embodiments of the invention, the anti-EGFR antibodies orADCs described herein can be used in a combination therapy with aninhibitor of NAMPT (see examples of inhibitors in US 2013/0303509;AbbVie, Inc., incorporated by reference herein) to treat a subject inneed thereof. NAMPT (also known as pre-B-cell-colony-enhancing factor(PBEF) and visfatin) is an enzyme that catalyzes the phosphoribosylationof nicotinamide and is the rate-limiting enzyme in one of two pathwaysthat salvage NAD. In one embodiment of the invention, anti-EGFRantibodies and ADCs described herein are administered in combinationwith a NAMPT inhibitor for the treatment of cancer in a subject.

In particular embodiments of the invention, the anti-EGFR antibodies orADCs described herein can be used in a combination therapy with SN-38,which is the active metabolite of the topoisomerase inhibitoririnotecan.

In other embodiments of the invention, the anti-EGFR antibodies or ADCsdescribed herein can be used in a combination therapy with a PARP (polyADP ribose polymerase) inhibitor, e.g., veliparib, to treat cancer,including breast, ovarian and non-small cell lung cancers.

Further examples of additional therapeutic agents that can beco-administered and/or formulated with anti-EGFR antibodies or anti-EGFRADCs described herein, include, but are not limited to, one or more of:inhaled steroids; beta-agonists, e.g., short-acting or long-actingbeta-agonists; antagonists of leukotrienes or leukotriene receptors;combination drugs such as ADVAIR; IgE inhibitors, e.g., anti-IgEantibodies (e.g., XOLAIR®, omalizumab); phosphodiesterase inhibitors(e.g., PDE4 inhibitors); xanthines; anticholinergic drugs; mastcell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or itsreceptors including H1, H2, H3, and H4, and antagonists of prostaglandinD or its receptors (DP1 and CRTH2). Such combinations can be used totreat, for example, asthma and other respiratory disorders. Otherexamples of additional therapeutic agents that can be co-administeredand/or formulated with anti-EGFR antibodies or anti-EGFR ADCs describedherein, include, but are not limited to, one or more of, temozolomide,ibrutinib, duvelisib, and idelalisib. Additional examples of therapeuticagents that can be co-administered and/or formulated with one or moreanti-EGFR antibodies or fragments thereof include one or more of: TNFantagonists (e.g., a soluble fragment of a TNF receptor, e.g., p55 orp75 human TNF receptor or derivatives thereof, e.g., 75 kD TNFR-IgG (75kD TNF receptor-IgG fusion protein, ENBREL)); TNF enzyme antagonists,e.g., TNF converting enzyme (TACE) inhibitors; muscarinic receptorantagonists; TGF-beta antagonists; interferon gamma; perfenidone;chemotherapeutic agents, e.g., methotrexate, leflunomide, or a sirolimus(rapamycin) or an analog thereof, e.g., CCI-779; COX2 and cPLA2inhibitors; NSAIDs; immunomodulators; p38 inhibitors, TPL-2, MK-2 andNFkB inhibitors, among others.

Other preferred combinations are cytokine suppressive anti-inflammatorydrug(s) (CSAIDs); antibodies to or antagonists of other human cytokinesor growth factors, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-15, IL-16, IL-18, IL-21, IL-31, interferons, EMAP-II,GM-CSF, FGF, EGF, PDGF, and edothelin-1, as well as the receptors ofthese cytokines and growth factors. Antibodies of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA, CTLA-4, PD-1, ortheir ligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the inflammatory cascade; preferred examples include TNFantagonists like chimeric, humanized or human TNF antibodies,adalimumab, (HUMIRA; D2E7; PCT Publication No. WO 97/29131 and U.S. Pat.No. 6,090,382, incorporated by reference herein), CA2 (Remicade™), CDP571, and soluble p55 or p75 TNF receptors, derivatives, thereof,(p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also TNF convertingenzyme (TACE) inhibitors; similarly IL-1 inhibitors(Interleukin-1-converting enzyme inhibitors, IL-IRA etc.) may beeffective for the same reason. Other preferred combinations includeInterleukin 4.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an ADC, an antibody or antibodyportion of the invention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. Inone embodiment, the dose of the antibodies and ADCs described herein is1 to 6 mg/kg, including the individual doses recited therein, e.g., 1mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, and 6 mg/kg. In anotherembodiment, the dose of the antibodies and ADCs described herein is 1 to200 μg/kg, including the individual doses recited therein, e.g., 1μg/kg, 2 μg/kg, 3 μg/kg, 4 μg/kg, 5 μg/kg, 10 μg/kg, 20 μg/kg, 30 μg/kg,40 μg/kg, 50 μg/kg, 60 μg/kg, 80 μg/kg, 100 μg/kg, 120 μg/kg, 140 μg/kg,160 μg/kg, 180 μg/kg and 200 μg/kg. It is to be noted that dosage valuesmay vary with the type and severity of the condition to be alleviated.It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that dosageranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed composition.

In one embodiment, an anti-EGFR antibody described herein, e.g., AbA, oran antigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 0.1 to 30mg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 1 to 15mg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 1 to 10mg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 2 to 3.In another embodiment, the anti-EGFR antibody, e.g., AbA, or an antigenbinding portion thereof, is administered to a subject in need thereof,e.g., a subject having cancer, as an ADC at a dose of 1 to 4 mg/kg.

In one embodiment, an anti-EGFR antibody described herein, e.g., AbA, oran antigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 1 to 200μg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 5 to 150μg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 5 to 100μg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 5 to 90μg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 5 to 80μg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 5 to 70μg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 5 to 60μg/kg. In another embodiment, the anti-EGFR antibody, e.g., AbA, or anantigen binding portion thereof, is administered to a subject in needthereof, e.g., a subject having cancer, as an ADC at a dose of 10 to 80μg/kg.

In one embodiment, an anti-EGFR ADC described herein, e.g., AbA-vc-MMAE,is administered to a subject in need thereof, e.g., a subject havingcancer, at a dose of 0.1 to 6 mg/kg. In another embodiment, an anti-EGFRADC described herein, e.g., AbA-vc-MMAE, is administered to a subject inneed thereof, e.g., a subject having cancer, at a dose of 0.5 to 4mg/kg. In another embodiment, an anti-EGFR ADC described herein, e.g.,AbA-vc-MMAE, is administered to a subject in need thereof, e.g., asubject having cancer, at a dose of 1.8 to 2.4 mg/kg. In anotherembodiment, an anti-EGFR ADC described herein, e.g., AbA-vc-MMAE, isadministered to a subject in need thereof, e.g., a subject havingcancer, at a dose of 1 to 4 mg/kg. In another embodiment, an anti-EGFRADC described herein, e.g., AbA-vc-MMAE, is administered to a subject inneed thereof, e.g., a subject having cancer, at a dose of about 1 mg/kg.In another embodiment, an anti-EGFR ADC described herein, e.g.,AbA-vc-MMAE, is administered to a subject in need thereof, e.g., asubject having cancer, at a dose of 3 to 6 mg/kg. In another embodiment,an anti-EGFR ADC described herein, e.g., AbA-vc-MMAE, is administered toa subject in need thereof, e.g., a subject having cancer, at a dose of 3mg/kg. In another embodiment, an anti-EGFR ADC described herein, e.g.,AbA-vc-MMAE, is administered to a subject in need thereof, e.g., asubject having cancer, at a dose of 2 to 3 mg/kg. In another embodiment,an anti-EGFR ADC described herein, e.g., AbA-vc-MMAE, is administered toa subject in need thereof, e.g., a subject having cancer, at a dose of 6mg/kg.

In another embodiment, an anti-EGFR antibody described herein,conjugated to a drug, e.g., a PBD, (an ADC) is administered to a subjectin need thereof, e.g., a subject having cancer, at a dose of 1 to 200μg/kg. In another embodiment, an anti-EGFR ADC described herein, isadministered to a subject in need thereof, e.g., a subject havingcancer, at a dose of 5 to 100 μg/kg. In another embodiment, an anti-EGFRADC described herein, is administered to a subject in need thereof,e.g., a subject having cancer, at a dose of 5 to 90 μg/kg. In anotherembodiment, an anti-EGFR ADC described herein, is administered to asubject in need thereof, e.g., a subject having cancer, at a dose of 5to 80 μg/kg. In another embodiment, an anti-EGFR ADC described herein,is administered to a subject in need thereof, e.g., a subject havingcancer, at a dose of 5 to 70 μg/kg. In another embodiment, an anti-EGFRADC described herein, is administered to a subject in need thereof,e.g., a subject having cancer, at a dose of 5 to 60 μg/kg.

Doses described above may be useful for the administration of eitheranti-EGFR ADCs or antibodies disclosed herein.

In another aspect, this application provides a method for detecting thepresence of EGFR in a sample in vitro (e.g., a biological sample, suchas serum, plasma, tissue, biopsy). The subject method can be used todiagnose a disorder, e.g., a cancer. The method includes: (i) contactingthe sample or a control sample with the anti-EGFR antibody or fragmentthereof as described herein; and (ii) detecting formation of a complexbetween the anti-EGFR antibody or fragment thereof, and the sample orthe control sample, wherein a statistically significant change in theformation of the complex in the sample relative to the control sample isindicative of the presence of EGFR in the sample.

Given their ability to bind to human EGFR, the anti-human EGFRantibodies, or portions thereof, of the invention, (as well as ADCsthereof) can be used to detect human EGFR (e.g., in a biological sample,such as serum or plasma), using a conventional immunoassay, such as anenzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) ortissue immunohistochemistry. In one aspect, the invention provides amethod for detecting human EGFR in a biological sample comprisingcontacting a biological sample with an antibody, or antibody portion, ofthe invention and detecting either the antibody (or antibody portion)bound to human EGFR or unbound antibody (or antibody portion), tothereby detect human EGFR in the biological sample. The antibody isdirectly or indirectly labeled with a detectable substance to facilitatedetection of the bound or unbound antibody. Suitable detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Alternative to labeling the antibody, human EGFR can be assayed inbiological fluids by a competition immunoassay utilizing rhEGFRstandards labeled with a detectable substance and an unlabeledanti-human EGFR antibody. In this assay, the biological sample, thelabeled rhEGFR standards and the anti-human EGFR antibody are combinedand the amount of labeled rhEGFR standard bound to the unlabeledantibody is determined. The amount of human EGFR in the biologicalsample is inversely proportional to the amount of labeled rhEGFRstandard bound to the anti-EGFR antibody. Similarly, human EGFR can alsobe assayed in biological fluids by a competition immunoassay utilizingrhEGFR standards labeled with a detectable substance and an unlabeledanti-human EGFR antibody.

In yet another aspect, this application provides a method for detectingthe presence of EGFR in vivo (e.g., in vivo imaging in a subject). Thesubject method can be used to diagnose a disorder, e.g., aEGFR-associated disorder. The method includes: (i) administering theanti-EGFR antibody or fragment thereof as described herein to a subjector a control subject under conditions that allow binding of the antibodyor fragment to EGFR; and (ii) detecting formation of a complex betweenthe antibody or fragment and EGFR, wherein a statistically significantchange in the formation of the complex in the subject relative to thecontrol subject is indicative of the presence of EGFR.

VI. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising anantibody, or antigen binding portion thereof, or ADC of the inventionand a pharmaceutically acceptable carrier. The pharmaceuticalcompositions comprising antibodies or ADCs of the invention are for usein, but not limited to, diagnosing, detecting, or monitoring a disorder,in preventing, treating, managing, or ameliorating of a disorder or oneor more symptoms thereof, and/or in research. In a specific embodiment,a composition comprises one or more antibodies of the invention. Inanother embodiment, the pharmaceutical composition comprises one or moreantibodies or ADCs of the invention and one or more prophylactic ortherapeutic agents other than antibodies or ADCs of the invention fortreating a disorder in which EGFR activity is detrimental. Preferably,the prophylactic or therapeutic agents known to be useful for or havingbeen or currently being used in the prevention, treatment, management,or amelioration of a disorder or one or more symptoms thereof. Inaccordance with these embodiments, the composition may further compriseof a carrier, diluent or excipient.

The antibodies and antibody-portions or ADCs of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion or ADC.

In one embodiment, the invention features a lyophilized formulationcomprising an anti-EGFR antibody drug conjugate, sucrose, polysorbate80, and histidine, wherein the formulation has a pH of about 5-7,wherein the anti-EGFR antibody drug conjugate comprises an anti-EGFRantibody, or antigen binding portion thereof, conjugated tomonomethylauristatin E (MMAE). In one embodiment, the invention furtherprovides a lyophilized formulation comprising an anti-EGFR ADCcomprising an anti-EGFR antibody, or antigen-binding portion thereof, asdescribed herein, conjugated to an auristatin, e.g., MMAE, a sugar,e.g., sucrose, a surfactant, e.g., a polysorbate, such as polysorbate80, and histidine. In one embodiment, the lyophilized formulationcomprises 1-20 mg of histidine, about 320-410 mg of a sugar, about 0.1to 0.9 mg of a surfactant, and about 1-150 mg of an anti-EGFR ADCcomprising an anti-EGFR antibody, or antigen-binding portion thereof, asdescribed herein, conjugated to an auristatin, e.g., MMAE. The inventionalso provides an aqueous formulation comprising about 1-100 mg/ml of ananti-EGFR ADC comprising an anti-EGFR antibody, or antigen-bindingportion thereof, as described herein, conjugated to an auristatin, e.g.,MMAE, about 1-10 mg/mL histidine, about 50-90 mg/ml of a sugar, e.g.,sucrose, and about 0.01-0.2 mg/ml of a surfactant, e.g., polysorbate 80.

Various delivery systems are known and can be used to administer one ormore antibodies or ADCs of the invention or the combination of one ormore antibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration can be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934, 272, 5,874,064,5,855,913, 5,290, 540, and 4,880,078; and PCT Publication Nos. WO92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, eachof which is incorporated herein by reference their entireties. In oneembodiment, an antibody of the invention, combination therapy, or acomposition of the invention is administered using Alkermes AIR®pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than anantibody of the invention of a subject to prevent, treat, manage, and/orameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent of theinvention can be delivered in a controlled release or sustained releasesystem. In one embodiment, a pump may be used to achieve controlled orsustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref.Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek etal., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymericmaterials can be used to achieve controlled or sustained release of thetherapies of the invention (see e.g., Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983,J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howardet al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat.No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT PublicationNo. WO 99/20253. Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferredembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. In yet another embodiment, a controlled or sustainedrelease system can be placed in proximity of the prophylactic ortherapeutic target, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human ColonCancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gel caps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985, 320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; andPCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346,and WO 99/66903, each of which is incorporated herein by reference theirentireties. In a specific embodiment, an antibody of the invention,combination therapy, and/or composition of the invention is administeredusing Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, at least 10 mg, at least 15 mg, at least25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg,or at least 100 mg. The lyophilized prophylactic or therapeutic agentsor pharmaceutical compositions of the invention should be stored atbetween 2° C. and 8° C. in its original container and the prophylacticor therapeutic agents, or pharmaceutical compositions of the inventionshould be administered within 1 week, within 5 days, within 72 hours,within 48 hours, within 24 hours, within 12 hours, within 6 hours,within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, one or more of theprophylactic or therapeutic agents or pharmaceutical compositions of theinvention is supplied in liquid form in a hermetically sealed containerindicating the quantity and concentration of the agent. Preferably, theliquid form of the administered composition is supplied in ahermetically sealed container at least 0.25 mg/ml, at least 0.5 mg/ml,at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid formshould be stored at between 2° C. and 8° C. in its original container.

The antibodies and antibody-portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. Preferably, the antibody or antibody-portions will beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 2-4%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants. The pharmaceutical composition comprising theantibodies and antibody-portions of the invention prepared as aninjectable solution for parenteral administration, can further comprisean agent useful as an adjuvant, such as those used to increase theabsorption, or dispersion of a therapeutic protein (e.g., antibody). Aparticularly useful adjuvant is hyaluronidase, such as Hylenex®(recombinant human hyaluronidase). Addition of hyaluronidase in theinjectable solution improves human bioavailability following parenteraladministration, particularly subcutaneous administration. It also allowsfor greater injection site volumes (i.e. greater than 1 ml) with lesspain and discomfort, and minimum incidence of injection site reactions.(see WO2004078140, US2006104968 incorporated herein by reference).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies and antibody-portions or ADCs of the invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection, intravenous injection or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, an antibody or antibody portion or ADC of theinvention may be orally administered, for example, with an inert diluentor an assimilable edible carrier. The compound (and other ingredients,if desired) may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into thesubject's diet. For oral therapeutic administration, the compounds maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

In other embodiments, an antibody or antibody portion or ADC of theinvention may be conjugated to a polymer-based species such that saidpolymer-based species may confer a sufficient size upon said antibody orantibody portion of the invention such that said antibody or antibodyportion of the invention benefits from the enhanced permeability andretension effect (EPR effect) (See also PCT Publication No.WO2006/042146A2 and U.S. Publication Nos. 2004/0028687A1,2009/0285757A1, and 2011/0217363A1, and U.S. Pat. No. 7,695,719 (each ofwhich is incorporated by reference herein in its entirety and for allpurposes).

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion orADC of the invention is formulated with and/or co-administered with oneor more additional therapeutic agents that are useful for treatingdisorders in which EGFR activity is detrimental. For example, ananti-hEGFR antibody or antibody portion or ADC of the invention may beformulated and/or co-administered with one or more additional antibodiesthat bind other targets (e.g., antibodies that bind cytokines or thatbind cell surface molecules). Furthermore, one or more antibodies of theinvention may be used in combination with two or more of the foregoingtherapeutic agents. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies.

In certain embodiments, an antibody or ADC to EGFR or fragment thereofis linked to a half-life extending vehicle known in the art. Suchvehicles include, but are not limited to, the Fc domain, polyethyleneglycol, and dextran. Such vehicles are described, e.g., in U.S.application Ser. No. 09/428,082 and published PCT Application No. WO99/25044, which are hereby incorporated by reference for any purpose.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the invention in detail, the samewill be more clearly understood by reference to the following examples,which are included for purposes of illustration only and are notintended to be limiting

EXAMPLES Example 1: Identification of Improved Anti-EGFR AntibodiesAntibody 1

Antibody 1 (Ab1) is a humanized anti-EGFR antibody. The heavy chainvariable region (VH) amino acid sequence of Ab1 is provided below as SEQID NO: 1. The VH CDR amino acid sequences of Ab1 are underlined belowand are as follows: GYSISSDFAWN (VH CDR1; SEQ ID NO: 2);YISYSGNTRYQPSLKS (VH CDR2; SEQ ID NO: 3); and AGRGFPY (VH CDR3; SEQ IDNO: 4).

Ab1 VH sequence (SEQ ID NO: 1)QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAG RGFPYWGQGTLVTVSSThe light chain variable region (VL) amino acid sequence of Ab1 isprovided below as SEQ ID NO 5. The VL CDR amino acid sequences of Ab1are underlined below and are as follows: HSSQDINSNIG (VL CDR1; SEQ IDNO: 6); HGTNLDD (VL CDR2; SEQ ID NO: 7); and VQYAQFPWT (VL CDR3; SEQ IDNO: 8).

Ab1 VL sequence (SEQ ID NO: 5)DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTF GGGTKLEIK

A screen was performed to identify anti-EGFR antibodies having improvedproperties over Ab1. The details of the identification of Ab1 variantsare provided below.

Preparation of Ab1 variant VL and VH libraries

Ab1 variant antibodies were identified by screening three single chain(scFv) libraries containing variant heavy or light chain variableregions of Ab1. One of the scFv libraries contained Ab1 variant variableheavy chains (i.e., scFvs containing an Ab1 variant variable heavy chainand an Ab1 variable light chain), while the second scFv librarycontained Ab1 variant variable light chains (i.e., scFvs containing anAb1 variant variable light chain and an Ab1 variable heavy chain). Ab1variant VH and VL regions were also combined into a third scFv librarywhich was subsequently screened through multiple rounds to selectcombinations of VH and VL Ab1 variants having higher binding affinitythan Ab1. The design of the Ab1 variant VH and VL libraries is shown inFIG. 16.

To create variant Ab1 VH regions, twelve amino acid residues wereselected for mutation based, at least in part, on alignment of the VH ofAb1 with the VH4-4 (SEQ ID NO: 93) and VH4-b germline sequences (seeFIG. 16A where mutated amino acid residues are designated “X”). A P101Dalteration (designated “Z” in FIG. 16A) was also generated based onhuman germline and Ab1 sequences. Using the codon usage described above,it was calculated that by targeting twelve residues (“X” residues inFIG. 16A) a 10⁹ library would provide the majority of VH Ab1 variantswith four or fewer mutated residues per clone. As described in FIG. 16A,one framework residue (Q1E) was also changed to prevent N-terminalpyroglutamate formation.

To create variant Ab1 VL regions, eleven amino acid residues wereselected for mutation based, at least in part, on alignment of the VLregion of Ab1 with the IGKV1-12 (L5) germline sequence (see residuesmarked “X” in FIG. 16B) (IGKV1-12 is described in SEQ ID NO: 94).Residues at positions 33 and 52 (marked “1” and “2”, respectively, inFIG. 16B) were designed to have limited diversity. Residues at position33 were limited to L, V, I, or F, and residues at position 52 werelimited to S, A, T, and P.

Yeast libraries based on the foregoing were created using transformationmethods described in Benatuil et al. (2010) Protein Eng. Design, andSelection, 23 (4): 155-159.

Screening of scFv Ab1 Variant Libraries

Single chain variable fragments (scFvs) containing either the Ab1variant VH or VL regions were expressed from the respective libraries,and were screened based on the scFv's ability to bind truncated wildtype (wt) human EGFR1-525, and mutant EGFR(CA). EGFR (CA) is an EGFRvariant that contains cysteine to alanine mutations at positions 295 and307 (see Garrett et al. (2009) PNAS USA 106(13): 5082-5087). Librariescontaining about 1×10⁹ clones were screened.

Given that Ab1 binds to EGFR (CA) but does not substantially bindwtEGFR, initial rounds of screening used EGFR (CA) to identify Ab1variant heavy and light chains having improved binding affinity (k_(on),k_(off), or both rates) for EGFR(CA) relative to Ab1. Later rounds ofscreening, however, used EGFR(1-525) to identify Ab1 variants havingimproved binding affinity (k_(on), k_(off), or both rates) over Ab1 forEGFR(1-525).

Library screening was performed using two methods, including magneticbead sorting (MACS (magnetic cell separation technology)) and a FACSbased assay (for MACS and FACS see, e.g., Chao et al (2006) NatureProtocols 1:755-765; Feldhaus et al. (2003) Nature Biotech 21:163-170;and VanAntwerp (2000) Biotechnol. Prog. 16:31-37). At least two roundsof screening based on magnetic bead enrichment was performed followed byat least three rounds of screening based on flow cytometric sorting(Feldhaus and Siegel, Ch. 17 of Flow Cytometry Protocols, 2^(nd) ed.,ed. Hawley and Hawley, vol. 263). Both equilibrium and k_(off) selectionwere used to identify scFvs having improved binding over Ab1. In total,three libraries were screened for Ab1 variant VH and VL regions havingimproved binding over Ab1. Four to five rounds of screening wasperformed using the Ab1 variant VH and VL libraries, and five to sixrounds was performed for the combined Ab1 variant VH/VL library.Screening of the Ab1 variant VL library, the Ab1 variant VH library, andthe combined Ab1 variant VH and VL library resulted in theidentification of scFvs containing Ab1 variant VH and VL regions havinghigher binding affinities for EGFR (EGFR(1-525) and EGFR (CA)) over Ab1.

Identification of Improved Antibodies

Fifteen Ab1 variant scFvs identified as having improved bindingcharacteristics, including specific binding to EGFR(1-525), wereselected for conversion into IgG proteins (specifically IgG1antibodies). The fifteen Ab1 variant antibodies are described herein,and include Antibody A (referred to throughout as “AbA”) (see VH SEQ IDNO: 9; VL SEQ ID NO: 5), Antibody B (referred to herein as “AbB”) (seeVH SEQ ID NO: 64; VL SEQ ID NO: 65), Antibody C (referred to herein as“AbC”) (see VH SEQ ID NO: 66; VL SEQ ID NO: 67), and Antibody D(referred to herein as “AbD”) (see VH SEQ ID NO: 68; VL SEQ ID NO: 69),Antibody E (referred to herein as “AbE”) (see VH SEQ ID NO: 50; VL SEQID NO: 51), Antibody F (referred to herein as “AbF”) (see VH SEQ ID NO:52; VL SEQ ID NO: 53), Antibody G (referred to herein as “AbG”) (see VHSEQ ID NO: 72; VL SEQ ID NO: 73), Antibody H (referred to herein as“AbH”) (see VH SEQ ID NO: 54; VL SEQ ID NO: 55), Antibody J (referred toherein as “AbJ”) (see VH SEQ ID NO: 56; VL SEQ ID NO: 57), Antibody K(referred to herein as “AbK”), Antibody L (referred to herein as “AbL”)(see VH SEQ ID NO: 58; VL SEQ ID NO: 59), Antibody M (referred to hereinas “AbM”) (see VH SEQ ID NO: 76; VL SEQ ID NO: 77), Antibody N (referredto herein as “AbN”) (see VH SEQ ID NO: 60; VL SEQ ID NO: 61), Antibody 0(referred to herein as “AbO”) (see VH SEQ ID NO: 62; VL SEQ ID NO: 63),and Antibody P (referred to herein as “AbP”) (see VH SEQ ID NO: 78; VLSEQ ID NO: 79). Selected clones from the VH and VL libraries were pairedwith the Ab1 VL or VH region, respectively (see AbA, AbB, AbC, AbD, AbE,and AbF).

Amino acid sequences of the VH regions of the Ab1variant antibodies are provided below. The CDRsare underlined, and the amino acid changesrelative to Ab1 are highlighted in bold. AbA VH (SEQ ID NO: 9)EVQLQESGPGLVKPSQTLSLTCTVSGYSISRDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPYWGQGTLVTVSSAbB VH (SEQ ID NO: 64)EVQLQESGPGLVKPSQTLSLTCTVSGYSISNDFAWNWIRQPPGKGLEWMGYISYKGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPW WGQGTLVTVSSAbC VH (SEQ ID NO: 66)EVQLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAG RGFPYWGQGTLVTVSSAbD VH (SEQ ID NO: 68)EVQLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAG RGFPYWGQGTLVTVSSAbE VH (SEQ ID NO: 50)EVQLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAG RGFPYWGQGTLVTVSSAbF VH (SEQ ID NO: 52)EVQLQESGPGLVKPSQTLSLTCTVSGYSISRDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPYWGQGTLVTVSSAbG VH (SEQ ID NO: 72)EVQLQESGPGLVKPSQTLSLTCTVSGYSISNDFAWNWIRQLPGKGLEWMGYISYKGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGLPYWGQGTLVTVSSAbH VH (SEQ ID NO: 54)EVQLQESGPGLVKPSQTLSLTCTVSGYSIGKDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGLPYWGQGTLVTVSSAbJ VH (SEQ ID NO: 56)EVQLQESGPGLVKPSQTLSLTCTVSGYSIGKDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGLPYWGQGTLVTVSSAbK VH (SEQ ID NO: 74)EVQLQESGPGLVKPSQTLSLTCTVSGYSISRDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPW WGQGTLVTVSSAbL VH (SEQ ID NO: 58)EVQLQESGPGLVKPSQTLSLTCTVSGYSIGKDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGLPYWGQGTLVTVSSAbM VH (SEQ ID NO: 76)EVQLQESGPGLVKPSQTLSLTCTVSGYSIGRDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPYWGQGTLVTVSSAbN VH (SEQ ID NO: 60)EVQLQESGPGLVKPSQTLSLTCTVSGYSIGRDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPYWGQGTLVTVSSAbO VH (SEQ ID NO: 62)EVQLQESGPGLVKPSQTLSLTCTVSGYSIGKDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPYWGQGTLVTVSSAbQ VH (SEQ ID NO: 70)EVQLQESGPGLVKPSQTLSLTCTVSGYSISHDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS WGLPW WGQGTLVTVSSAbP VH (SEQ ID NO: 78)EVQLQESGPGLVKPSQTLSLTCTVSGYSISHDFAWNWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS WGLPW WGQGTLVTVSSAmino acid sequences of the VL regions of the Ab1variant antibodies are provided below. The CDRsare underlined, and the amino acid changesrelative to Ab1 are highlighted in bold. AbA VL (SEQ ID NO: 5)DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTF GGGTKLEIK AbB VL(SEQ ID NO: 65) DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTF GGGTKLEIK AbC VL(SEQ ID NO: 67) DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYEQFPWTF GGGTKLEIK AbD VL(SEQ ID NO: 69) DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNLGWLQQKPGKSFKGLIYHGANLHDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAEFPWTF GGGTKLEIK AbE VL(SEQ ID NO: 51) DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNLGWLQQKPGKSFKGLIYHGSNLDH GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDQFPWTF GGGTKLEIK AbF VL(SEQ ID NO: 53) DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAQFPWTF GGGTKLEIK AbG VL(SEQ ID NO: 73) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNIGWLQQKPGKSFKGLIYHGANLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDEFPWTF GGGTKLEIK AbH VL(SEQ ID NO: 55) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNIGWLQQKPGKSFKGLIYHGANLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDEFPWTF GGGTKLEIK AbJ VL(SEQ ID NO: 57) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNIGWLQQKPGKSFKGLIYHGANLDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDEFPWTF GGGTKLEIK AbK VL(SEQ ID NO: 75) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNVGWLQQKPGKSFKGLIYHGSNLDH GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDDFPWTF GGGTKLEIK AbL VL(SEQ ID NO: 59) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNVGWLQQKPGKSFKGLIYHGSNLDH GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDDFPWTF GGGTKLEIK AbM VL(SEQ ID NO: 77) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNVGWLQQKPGKSFKGLIYHGSNLDH GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDDFPWTF GGGTKLEIK AbN VL(SEQ ID NO: 61) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNVGWLQQKPGKSFKGLIYHGSNLDH GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDDFPWTF GGGTKLEIK AbO VL(SEQ ID NO: 63) DIQMTQSPSSMSVSVGDRVTITCHSSQDITYNVGWLQQKPGKSFKGLIYHGSNLDH GVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYDDFPWTF GGGTKLEIK AbQ VL(SEQ ID NO: 71) DIQMTQSPSSMSVSVGDRVTITCHSSQDINMNVGWLQQKPGKSFKGLIYHGAILDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAEFPWTF GGGTKLEIK AbP VL(SEQ ID NO: 79) DIQMTQSPSSMSVSVGDRVTITCHSSQDINMNVGWLQQKPGKSFKGLIYHGAILDDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYAEFPWTF GGGTKLEIK

Nucleic acid sequences of the fifteen Ab1 variant VH and/or VL domainswere subcloned into expression vectors for expression of full length IgGantibodies (see vectors and methods disclosed in U.S. Pat. No. 8,187,836and U.S. Pat. No. 8,455,219, both incorporated by reference herein). Theantibody expression vectors were transiently transfected into HEK293cells according to standard methods (see Durocher et al. (2002) NucleicAcid Res. 30(2;e9)). The amino acid sequence of the leader sequence usedfor expression of the heavy chain of each of the Ab1 variants wasMEFGLSWLFLVAILKGVQC (SEQ ID NO: 88), while the amino acid sequence usedfor the leader sequence for the expression of the light chain of each ofthe Ab1 variants was MRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 89). The Ab1antibody variants were subsequently purified from media by protein Achromatography for affinity and functional assessment.

Antibody AbA

One of the identified Ab1 variant antibodies was AbA. AbA has the samevariable light chain sequence as Ab1 (SEQ ID NO: 5), including the sameCDR1, CDR2, and CDR3 amino acid sequences (described in SEQ ID NOs: 6,7, and 8, respectively).

The VH amino acid sequence of AbA is provided below in SEQ ID NO: 9. TheVH CDR amino acid sequences of AbA are as follows: GYSISRDFAWN (CDR1;SEQ ID NO: 10); YISYNGNTRYQPSLKS (CDR2; SEQ ID NO: 11); and ASRGFPY(CDR3; SEQ ID NO: 12), and are underlined below. Residues that aredifferent in the heavy chain variable region of AbA versus Ab1 are shownbelow in bold.

AbA VH amino acid sequence (SEQ ID NO: 9)EVQLQESGPGLVKPSQTLSLTCTVSGYSISRDFAWNWIRQPPGKGLEWMGYISYNGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTAADTATYYCVTAS RGFPYWGQGTLVTVSS

FIGS. 1 and 2 provide an alignment of the amino acid sequences of the VHand VL regions (FIG. 1) and the complete heavy and light chains (FIG. 2)of Ab1 and AbA. The light chain amino acid sequences of Ab1 and AbA arethe same (SEQ ID NO: 13). The heavy chain amino acid sequences of Ab1and AbA, however, have six amino acid differences between the twosequences, three of which are in the CDRs. Differences between the Ab1VH amino acid sequence and the AbA VH amino acid sequence are shaded inFIG. 1 and are found in each of the VH CDRs. The CDR1 domain of thevariable heavy chain of AbA included an amino acid change from a serine(Ab1) to an arginine. The CDR2 domain of the variable heavy chainincluded an amino acid change from a serine in Ab1 to an asparagine inAbA. Finally, the CDR3 domain of the variable heavy chain included anamino acid change from a glycine in Ab1 to a serine in AbA. Two of theamino acid changes within AbA are in the constant region of the heavychain (D354E and L356M). The Fc region amino acid mutations in AbArepresent human IgG allotype changes from a z, a allotype to a z, non-aallotype. In addition to the other changes, the first amino acid waschanged from a glutamine (Q) to a glutamic acid (E), as described, forexample, in FIG. 1.

Comparison of Ab1 Variant Antibody Sequences to Ab1 Sequence

Table 1 provides an alignment of the amino acid sequences of the heavyand light chain CDRs for Ab1 variant antibodies AbA, AbG, AbK, AbM, andAbP in comparison to Ab1. Table 2 provides a comparison of the anti-EGFRantibody CDR consensus sequences for Ab1, AbA, AbG, AbK, AbM, and AbP.Blank spaces in Tables 1 and 3 indicate that the residue is the same asAb1.

TABLE 1Heavy and Light Chain CDR Sequence Comparison of Ab1 vs. AbA, AbG, AbK,AbM, and AbP Variants HEAVY CHAIN CDRS SEQ SEQ SEQ Variable Heavy ChainID VH  ID ID (VH) CDR1 NO: CDR2 NO: VH CDR3 NO: Ab1 G Y S I S S D F A WN 2 Y I S Y S G N T R Y Q P S L K S 3 A G R G F P Y 4 AbA R 10 N 11 S 12AbG N 16 K 17 S L 18 AbK R 10 N 11 S W 19 AbM G R 20 N 11 S 12 AbP H 213 S W L W 22 LIGHT CHAIN CDRS SEQ SEQ SEQ Variable Light Chain ID ID ID(VL) CDR1 NO: VL CDR2  NO: VL CDR3  NO: Ab1 H S S Q D I N S N I G 6 H GT N L D D 7 V Q Y A Q F P W T 8 AbA 6 7 8 AbG T Y 23 A 24 D E 25 AbK T YV 26 S H 27 D D 28 AbM T Y V 26 S H 27 D D 28 AbP M V 29 A I 30 E 31

TABLE 2 CDR Consensus Sequences for Ab1 Variants from Table 1 CDR SEQ IDCDR Consensus Sequences for Ab1 region NO: Variants VH CDR1 SEQ IDG Y S I(S/G/H) (S/R/N)D F A W N NO: 35 VH CDR2 SEQ IDY I S Y(S/N/K)G N T R Y Q P S L K S NO: 36 VH CDR3 SEQ ID A  S(R/W)G(F/L)P(Y/W) NO: 37 VL CDR1 SEQ ID H S S Q D I(N/T) (Y/M/S)N(I/V)GNO: 38 VL CDR2 SEQ ID H G (T/A/S) (N/I)L D(D/H) NO: 39 VL CDR3 SEQ IDV Q Y(A/D) (Q/E/D)F P W T NO: 40

As described in Table 2, the Ab1 variant antibodies AbA, AbG, AbK, AbM,AbP each has a serine residue in the variable heavy chain of CDR3 inplace of a glycine (shown in bold/underlined in Table 2).

A comparison of the VH and VL CDR sequences of Ab1 versus antibodiesAbB, AbC, AbD, AbE, AbF, AbH, AbJ, AbL, AbN, AbO, and AbQ is describedin Table 3. In addition to the CDR changes described in Table 3, AbG hasan amino acid residue change within the framework 2 regions of the VH.

TABLE 3Heavy and Light Chain CDR Sequence Comparison of Ab1 vs. Certain Ab1 VariantsHEAVY CHAIN CDRS SEQ SEQ SEQ Variable Heavy Chain ID ID ID (VH) CDR1 NO:VH CDR2 NO: VH CDR3 NO: Ab1 G Y S I S S D F A W N 2 Y I S Y S G N T R YQ P S L K S 3 A G R G F P Y 4 AbB N 16 K 17 S W 19 AbC 2 3 4 AbD 2 3 4AbE 2 3 4 AbF R 10 3 S 12 AbH G K 80 N 11 S L 18 AbJ G K 80 3 S L 18 AbLG K 80 N 11 S L 18 AbN G R 20 3 S 12 AbO G K 80 N 11 S 12 AbQ H 81 N 11S W L W 22 LIGHT CHAIN CDRS SEQ SEQ SEQ Variable Light Chain ID ID ID(VL) CDR1 NO: VL CDR2 NO: VL CDR3 NO: Ab1 H S S Q D I N S N I G 6 H G TN L D D 7 V Q Y A Q F P W T 8 AbB 6 7 8 AbC 6 7 E 84 AbD L 82 A H 83 E31 AbE L 82 S H 27 D 85 AbF 6 7 8 AbH T Y 23 A 24 D E 25 AbJ T Y 23 A 24D E 25 AbL T Y V 26 S H 27 D D 28 AbN T Y V 26 S H 27 D D 28 AbO T Y V26 S H 27 D D 28 AbQ M V 29 A I 30 E 31

Characterization of the Ab1 variant antibodies are described in Examples2 to 8 below.

Example 2: Binding Analysis of Anti-EGFR Antibodies Biacore Analysis

Biacore analysis was performed to compare the affinity of Ab1, Ab2 (anantibody having the same six CDR amino acid sequences of cetuximab), andthe anti-EGFR antibodies identified in Example 1 to three forms ofrecombinant EGFR, specifically the wild type EGFR extra-cellular domain(ECD) (EGFR 1-645) (SEQ ID NO: 34), EGFRvIII (EGFR (1-29)-G-(298-645)(SEQ ID NO: 46)) and a truncated wild type EGFR 1-525 (EGFR1 (1-525)(SEQ ID NO: 47)). Ab2 includes the heavy and light chain amino acidsequences of Ab2 as provided in SEQ ID NOs: 48 and 49, respectively, andwas made according to standard methods.

While Ab1 and Ab2 are both anti-EGFR antibodies, they have distinctproperties and bind to unique epitopes. Ab2 binds to the L2 domain ofEGFR (Gan et al. (2012) Cancer Res 72(12)1-7; Li et al. (2005) CancerCell 7:301), whereas Ab1 binds to amino acid residues 287-302 of the CR1domain (domain II) of EGFR (Gan et al. (2012) 72(12)1-7; Johns et al.(2004) J Biol Chem 279:30375-30384). Domain II of EGFR is exposed in theextended conformation of EGFR (Li et al. (2005) Cancer Cell 7:301). FIG.17 provides a diagram of the overall structural domain organization ofEGFR and indicates generally where the epitopes for Ab2 and Ab1 arelocated. Unlike Ab2, Ab1 does not bind (or has very weak binding) to theEGFR ECD (SEQ ID NO: 34). Although Ab1 can bind to activated wild typeEGFR, and has a higher binding affinity for EGFRvIII versus Ab2 in vivo.Thus, Ab2 was used as a control in the experiments described herein as asecond anti-EGFR antibody that binds to a different epitope and hasdifferent binding affinity characteristics than Ab1.

Biacore analysis was performed using a Biacore T100 with a CMS sensorychip, and test antibodies were captured via anti-human Fc antibodiesthat were amino coupled to the chip using a standard amine coupling kitaccording to manufacturer's instructions (GE healthcare). Briefly, a CMSchip surface was activated with EDC/NHS. Goat anti-human Fc specificpolyclonal antibody (Thermo Fisher Scientific Inc., Rockford, Ill.) wasdiluted to 25 μg/mL in 10 mM sodium acetate (pH 4.5) and injected overthe activated surface to achieve immobilization. Unreacted moieties onthe biosensor surface were blocked with ethanolamine. The Biacore T100is a surface plasmon resonance based biosensor for detecting,characterizing and quantifying bimolecular interactions, including theinteractions between an antibody and its antigen. Antigen was injectedat 804/minute for 3 minutes and dissociation was followed for 15minutes. The EGFR ECD tested included amino acids 1-645 of EGFR fused toa myc and histidine tag (EGFR (1-645)-LESRGPF-Myc-NMHTG-6His (“LESRGPF”(SEQ ID NO: 90); “6His” (SEQ ID NO: 91)). The EGFRvIII variant was alsofused to myc and histidine tag (EGFR(1-29)-G-(298-645)-LESRGPF-Myc-NMHTG-6His), as was the ECD EGFR 1-525(EGFR1 (1-525)-SRGPF-Myc-NMHTG-6His (“SRGPF” (SEQ ID NO: 92)). Therunning buffer used in the Biacore analysis was HBS-EP+: 10 mM Hepes,pH7.5, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20.

Results from the Biacore binding analysis are shown in FIG. 3. Of theAb1 variant antibodies tested, AbK had the highest binding affinity fortruncated EGFR (1-525) with a K_(D) of 1.7×10⁻⁹ M, which was more than a1300 fold increase over the K_(D) of Ab1. As described in FIG. 3, AbEshowed the lowest determinable affinity for truncated EGFR (1-525) witha K_(D) of 5.9×10⁻⁷ M, only about a 4 fold increase in affinity over theK_(D) of Ab1. AbA showed a ten-fold improvement in K_(D) for truncatedEGFR (1-525) (K_(D) of 2.2×10⁻⁷ M (AbA) vs K_(D) of 2.3×10⁻⁶ M (Ab1)).The K_(D) ratios of AbB, AbC, AbD, AbF, AbG, AbH, AbJ, AbL, AbM, AbN,AbO, and AbP versus Ab1 are also described in FIG. 3 for truncated EGFR1-525.

Binding affinity of the Ab1 variant antibodies, Ab1, and Ab2 to EGFRvIIIwas also tested. As shown in FIG. 3, all of the Ab1 variants showedhigher binding affinity to EGFRvIII than Ab1. For example, AbAdissociated from EGFRvIII with a K_(D) of 2.3×10⁻⁹ M, whereas Ab1 had adissociation constant of K_(D) of 9.4×10⁻⁹ M. Thus, AbA had a four foldincrease in affinity (determined by dissociation constants) versus Ab1.

Ab1 variant antibodies that showed a higher dissociation constant fortruncated EGFR(1-525) relative to Ab1, did not necessarily show the sameincrease in affinity for EGFRvIII. For example, while AbO showed thegreatest fold increase (63 fold) in affinity for EGFRvIII relative toAb1, AbK showed the greatest fold increase (over 1300 fold increase) fortruncated EGFR(1-525) relative to Ab1, as described in FIG. 3. Inanother example, AbG showed the second highest fold increase in bindingto EGFRvIII among the Ab1 variant antibodies (with an increase of over47 fold), yet ranked sixth in the affinity increase for truncatedEGFR(1-525) relative to Ab1 (with a 263 fold increase), as described inFIG. 3.

Many of the Ab1 variants were determined in the Biacore testing ashaving lower affinity for both EGFR 1-525 and EGFRvIII than Ab2 whichhad, for example, a K_(D) of 4.0×10⁻⁹ M for truncated EGFR(1-525).

Biacore analysis revealed that neither Ab1 nor the Ab1 variantantibodies bound to the full length ECD of EGFR (EGFR amino acids1-645). In contrast, Ab2 bound to the ECD of EGFR. Thus, despite theobservation that binding of the Ab1 variants to the wild type EGFR ECDwas absent or negligible, as was also observed for Ab1, increasedbinding affinity to the truncated receptor EGFR(1-525) increased withthe Ab1 variants.

FACS (Fluorescence Activated Cell Sorting) Analysis

FACS analysis was performed to determine the binding characteristics ofAbA to tumor cells (A431 cells) in comparison to Ab1 and Ab2. Using FACSanalysis, the fluorescence intensity and cell count were determinedwhere the amount of antibody bound to the cells was reflected in thefluorescence intensity (i.e., the value of geometric mean) obtainedthrough analysis using flow cytometry software. Specifically, thebinding activity of the antibody, which is represented by the amount ofbound antibody, was be assessed by determining the value of thegeometric mean.

A431 cells were harvested from flasks at approximately 80% confluenceusing a cell dissociation buffer. The harvested A431 cells were washedonce in PBS/1% FBS (fetal bovine serum) (FACS buffer) then resuspendedat 2.5×10⁶ cells/mL in FACS buffer. 100 μL of cells/well were added to around bottom 96-well plate. 10 μL of a 10× concentration of antibody wasused (final concentrations are indicated in FIG. 4). Wells were washedtwice with FACS buffer and resuspended in 50 μL of secondary antibody(AlexaFluor 488) diluted in FACS buffer. The plate was incubated at 4°C. for one hour and washed twice with FACS buffer. Cells wereresuspended in 100 μL of PBS/1% formaldehyde and analyzed on a BectonDickinson LSRII flow cytometer. Data were analyzed using WinList flowcytometry analysis software.

Results from the FACS analysis of Ab1, Ab2, and AbA binding to A431tumor cells are provided in FIG. 4, which shows the geometric meanversus the concentration of antibody incubated with the cells. Asdescribed in FIG. 4, AbA had higher binding to EGFR on A431 tumor cellsthan Ab1, but AbA had a lower binding affinity in comparison to Ab2. Thedata in FIG. 4 represents direct binding of each antibody to itsantigen.

As described in FIGS. 3 and 4, AbA had increased binding affinity toEGFR and showed increased binding to cells with high levels of EGFR(A431 tumor cells) relative to Ab1. It was also determined that AbAexhibited a more modest increase in binding to cells with lower levelsof EGFR (data not shown), versus the binding to the A431 cells.

Example 3: Epitope Analysis of Anti-EGFR Antibodies

Tests were performed to determine whether the anti-EGFR antibodiesidentified in Example 1 had the same epitope as antibody Ab1 or whetherthe amino acid changes within Ab1 variant antibodies AbA, AbG, AbK, AbM,and AbP impacted epitope binding.

Competition Assay Analysis

A competition binding FACS assay was used to determine epitopespecificity among the improved anti-EGFR antibodies in comparison toAb1, Ab2, and Control 1 antibody (an anti-CD20 antibody (rituximab(Roche)) used as a negative control). U87MG cells (a human glioblastomacell line (obtained from A. Scott, Ludwig Institute for Cancer Research)(U87MG cells are available from ATTC as ATCC HTB-14™; see also, e.g.,U-87MG Cell Line human, Sigma-Aldrich) which express EGFRvIII were used.U87MG cells were harvested from flasks at approximately 80% confluenceusing a cell dissociation buffer. Cells were washed once in PBS/1% FBS(fetal bovine serum) (FACS buffer) then resuspended at 2.5×10⁶ cells/mLin FACS buffer. 100 μL of cells/well were added to a round bottom96-well plate. For competition FACS, Ab1 was fluorescently conjugated byaddition of fluorescein isothiocyanate (FITC) at a final concentrationof 100 nM (with or without the competing antibodies), and then wellswere washed twice in FACS buffer, suspended in 100 μL of PBS/1%formaldehyde, and analyzed on a Becton Dickinson LSRII flow cytometer.Fluorescence was measured at 488 nM.

The results from the competition assay are described in FIG. 5 andindicate that the Ab1 variant antibodies tested (i.e., AbA, AbG, AbK,AbM, and AbP) recognized the same epitope as Ab1 (domain II of EGFR,which is exposed in the “extended” EGFR conformation) given that the Geomean calculation of fluorescence decreased with an increase in unlabeledAb1 variant antibody concentration. The results also show that theAb1/AbA/AbG/AbK/AbM/AbP epitope is distinct from the Ab2 epitope, as nocompetition between Ab2 and Ab1 or between Ab2 and the Ab1 variantantibodies was observed. The Control 1 antibody also showed nodetectable binding to the Ab1 epitope in the competition assay, as theControl 1 antibody failed to compete with FITC labeled Ab1 for bindingto the cells.

Imaging Analysis

To evaluate the efficacy of antibody uptake by EGFR expressing tumors, aSPECT imaging assay was performed using AbA labeled with ¹¹¹In (see,e.g., Khalil, et al. International Journal of Molecular Imaging, Vol.2011, Article ID 796025, p. 1-15). Two tumor models, SW48 cells (colontumor cells; ATCC No. CCL-231™) and EBC-1 cells (EBC-1 cells are from ahuman lung squamous cell carcinoma line that is from the JapaneseResearch Resources Bank (Id No. RCB1965) (Tokyo, Japan)) were chosen fortheir moderate EGFR expression levels based on immunohistochemistryanalysis. Following injection of the tumor cells into the mice, andsubsequent administration of the labelled AbA antibody, labelled Ab1, orthe labelled negative control (a non-EGFR binding IgG), SPECT/CT imageswere acquired of the mice at 4, 12, 24, 48, 72, 120, and 168 hourspost-injection using a nanoSPECT/CT. All labelled antibodies were dosedvia venous tail injection. To account for differences in blood clearancefor the antibodies, data are reported as the ratio of tumor to blood.The results provided in both FIGS. 20A and 20B demonstrate higher AbAuptake into both the SW48 (FIG. 20A) and EBC-1 (FIG. 20B) cells in themouse compared to Ab1 and the IgG control. In the EBC1 model (FIG. 20B),which is the model with a lower level of EGFR expression, Ab1 uptake wassimilar to the IgG control while AbA uptake was higher than both Ab1 orthe negative control. The results provided in FIG. 20 demonstrate thatAbA is able to target specific EGFR-expressing tumors. Similar imagingresults for AbA were also observed using A431 tumor cells (derived froman epidermoid carcinoma solid tumor (ATCC No. CRL-1555) known to havehigh levels of EGFR expression.)

Example 4: In Vitro Analysis of Anti-EGFR Antibody Activity in TumorCell Lines In Vitro Analysis of EGFR Signaling in SCC-15 and H292 TumorCell Lines

The ability of Ab1, Ab2, AbA, AbB, AbC, AbD, AbG, AbK, AbM, and AbP toinhibit EGF-mediated tyrosine phosphorylation of EGFR in tumor celllines in vitro was assessed by Western blot analysis using squamouscarcinoma cells (SCC)-15 (ATCC® CRL-1623™) and H292 cells (lungcarcinoma cell line; ATCC® CRL-1848™). Both SCC-15 and H292 cellsexpress wild type EGFR. The down regulation of pEGFR in wild-type EGFRexpressing tumor cells induced by antibody treatment indicates thatthose antibodies function at least in part by inhibition of signalingthrough the receptor. SCC-15 cells (human tongue squamous cellcarcinoma) are transformed keratinocytes and are sensitive to Ab1 invivo. While SCC-15 cells are sensitive to Ab1 in vivo, H292 (humannon-small cell lung cancer (NSCLC)) cells are resistant to Ab1inhibition of EGF-mediated tyrosine phosphorylation of EGFR both invitro and in vivo. Thus, both cell lines were used to test the abilityof AbA, AbB, AbC, AbD, AbG, AbK, AbM, and AbP to inhibit EGFR signalingin tumor cells in vitro.

Cells were either plated at 60,000-80,000 per well in 24-well plate, orwere plated at 100,000-200,000 per well in 6-well tissue culture plates.Cells were incubated overnight in growth media. Followingserum-starvation for 24 hours at 37° C., where appropriate, cells wereincubated with the antibodies for one hour at 37° C., and thenstimulated with recombinant human EGF (R&D Systems) for 10 minutes at37° C. Cells were then washed twice with ice-cold PBS, and lysed with100-200 μL/well of Cell Lysis Buffer (Cell Signaling Technology)supplemented with Complete Mini Protease Inhibitor Cocktail (Roche) and0.1% NP40 (Tergitol-type NP-40; nonyl phenoxypolyethoxylethanol). Afterflash-freezing at −80° C. for at least 20 minutes, cell lysates werecleared by centrifugation at 14,000 rpm, for 10 minutes at 4° C. Proteinconcentrations of cleared sample lysates were determined via BCA ProteinAssay (Pierce_Thermo Scientific). Cell lysates (10 μg) were resolved bySDS-PAGE using 4-12% bis-Tris, Midi gels, (Life Technologies) andtransferred to nitrocellulose membranes using the iBlot Dry Transfersystem (Life Technologies). Blots were blocked with 5% milk/Tween-Trisbuffered saline (TTBS) for one hour at room temperature, washed threetimes with TTBS, and then incubated overnight with appropriate primaryantibodies (anti-Phosphotyrosine (4G10) biotin conjugate, Millipore,1:1000 dilution to detect phosphorylated EGFR; rabbit anti-EGFR,Lifespan Biosciences, 1:2000 dilution to detect total EGFR; rabbitanti-Pan actin, Cell Signaling Technology, 1:1000 dilution to detect theinternal control actin) at 4° C. Following overnight incubation withprimary antibodies, blots were washed three times with TTBS for fiveminutes, and then incubated for one hour at room temperature with eitherdonkey anti-rabbit antibody (Jackson Laboratories, 1:2000 dilution) todetect total EGFR and actin, or incubated with streptavidin-HRPconjugate (KPL, 1:1000 dilution) to detect phosphorylated EGFR. Blotswere then washed three times with TTBS, and treated with West DuraChemiluminescent substrate (Thermo Scientific). Blots were visualized byscanning using an LAS-4000 scanner (Fuji).

Ab2 was used as a positive control (i.e., an inhibitor of EGFRphosphorylation) and Control 1 antibody was used as a negative control(i.e., does not bind EGFR and, therefore, has no impact onphosphorylated EGFR).

Results from the Western blot analysis of the in vitro study usingSCC-15 cells are provided in FIG. 7A and indicate that Ab1 andantibodies AbA, AbB, AbC, and AbD were not able to significantly inhibitEGFR signaling in SCC-15 cells as indicated by no significant downregulation of phosphorylated EGFR. In contrast, a decrease inphosphorylated EGFR, as shown in FIG. 7A, indicated that antibodies AbG,AbK, AbP, and AbM decreased EGFR activity in SCC-15 cells in vitro giventhat the level of phosphorylated EGFR was lower relative to the negativecontrol and Ab1 and comparable to the positive control (Ab2) levels.Thus, a number of Ab1 variants gained in vitro activity versus Ab1.

Results from the Western blot analysis of the in vitro study using H292cells are provided in FIG. 7B. The results in FIG. 7B indicate that Ab1and antibodies AbA, AbB, AbC, and AbD showed little inhibition of EGFRsignaling in H292 cells in vitro with no significant reduction onphosphorylated EGFR in comparison to Ab2 and antibodies AbG, AbK, AbM,and AbP. Antibodies AbG, AbK, AbM, and AbP were more effective atinhibiting tyrosine phosphorylation of EGFR in H292 cells (see FIG. 7B)better than was Ab1. Ab2 (positive control) also inhibited EGFRphosphorylation, as described in FIG. 7B, whereas the negative control(Control 1 antibody) showed no detectable inhibition of phosphorylationgiven the results compared to Ab2.

As described below, while AbA was found to be relatively inactive atinhibiting EGFR phosphorylation in wild type EGFR positive cells (H292)in vitro, AbA was able to inhibit phosphorylation of these cells invivo, in comparison to antibody Ab1 tested under the same conditions.

Thus, the in vitro results described in FIGS. 7A and 7B indicate thatdespite the increased binding of AbA, AbB, AbC, AbD, AbG, AbK, AbM, andAbP to EGFR (relative to Ab1), only some of the Ab1 variant antibodies(i.e., AbG, AbK, AbM, and AbP) were able to significantly inhibit ordecrease EGF-mediated signaling in vitro in the tested tumor cell lines.Generally, the ability of the tested Ab1 variant antibodies to inhibitEGF-mediated tyrosine phosphorylation of EGFR in SCC-15 and H292 cellsin vitro correlated with increased affinity to both EGFRvIII andtruncated EGFR(1-525), as determined by surface plasmon resonance.

FIG. 6 provides a summary of the binding affinities of Ab1, Ab2, and theAb1 variant antibodies to EGFR(1-525). The two circle outlines in FIG. 6reflect the in vitro results described above (or results from similartests) with respect to the ability of the antibody to inhibit EGFRphosphorylation (indicating inhibition of EGFR activity) in either H292cells or SCC-15 cells. As described in FIG. 6, the Ab1 variantantibodies could be categorized into two groups (Groups 1 and 2): thosethat do not inhibit in vitro EGFR signaling in the tested tumor celllines based on the results provided in FIG. 7 (including Ab1, AbA, AbB,AbD, AbE, and AbF; designated Group 1 in FIG. 6), and those that doinhibit EGFR signaling in vitro in the tested tumor cell lines(including AbG, AbH, AbL, AbK, AbJ, AbM, AbN, AbO, and AbP; designatedGroup 2 in FIG. 6) as described in FIG. 7. The comparison provided inFIG. 6 indicates that all of the Ab1 variant antibodies had higheraffinity to EGFR(1-525) compared to Ab1, and that those with a K_(d) ofless than 1×10⁻⁴ s⁻¹ (Group 2) were able to significantly inhibitEGFR-signaling in vitro as described in the results presented in FIG. 7.As described in FIG. 6, the maturation process of Ab1 resulted primarilyin Ab1 variants having enhanced off-rates.

In Vitro Analysis of A431 Tumor Cell Line

The ability of Ab1 and the Ab1 variant antibodies to inhibitEGF-mediated phosphorylation of EGFR was also tested using A431 humanepithelial carcinoma cells using a phospho-EGFR ELISA assay. A431 cellsexpress wild type EGFR.

Cells were plated at 20,000 per well in collagen coated 96-well dishesin growth media. Twenty four hours later, cells were washed in serumfree media and serum starved for four hours. Where appropriate, cellswere pretreated with monoclonal antibody for one hour, and thenstimulated with recombinant EGF for 10 minutes at 37° C. Following EGFstimulation, cells were washed twice with ice-cold PBS, and lysed with100 pt/well of cell lysis buffer supplemented with protease inhibitorsand flash-frozen at −80° C. for at least 20 minutes. Capture plates weregenerated by pre-coating wells with 50 μL of an anti-EGFR antibody (R&Dsystems, part number 841402, at 0.8 μg/mL), followed by blocking withPBS/1% BSA treatment for one hour, and washed three times in Tween-Trisbuffered saline (TTBS). Cell lysates were added to capture plates andincubated at 4° C. overnight. Plates were washed five times in TTBS, andincubated with pTry-horse radish peroxidase (R&D Systems, DYC1095) forone hour. Plates were washed five times in TTBS and 100 μL of3,3,5,5-tetramethylbenzidine (TMB) was added to each well and incubatedat room temperature until color developed. Reactions were stopped byaddition of 1N HCl, and OD was read at 450 nm.

Results from the A431 inhibition study are described in FIGS. 8A and 8Band show that the Ab1 variant antibodies showed a range in their abilityto inhibit EGFR activity in A431 cells. As shown in FIG. 8A, Ab1 andvariants AbA, AbB, AbC, AbD, AbE, and AbF were ineffective at inhibitingEGFR signaling in A431 cells (determined by EGF-mediated phosphorylationof EGFR), even at concentrations of 1333 nM of antibody. In contrastAb1variant antibodies AbG, AbH, AbJ, AbK, AbL, AbM, AbN, AbO, AbP, andAbQ were more effective at blocking EGFR phosphorylation than Ab1,albeit at a higher concentration than Ab2. Control 1 antibody, whichdoes not bind to EGFR, had no effect on EGFR inhibition of A431 cells.

FIG. 8B expands on the data provided in FIG. 8A for antibodies Ab1, AbP,and Ab2. As described in FIG. 8B, Ab1 showed low levels of inhibition,i.e., an average of 10% or less, in comparison to Ab2 or AbP. While Ab1was determined to have an IC₅₀ value greater than 1333 nM at inhibitingEGFR signaling in A431 cells, AbP had an IC₅₀ value of greater than 40nM, which was an improvement over Ab1. Ab2 had an IC₅₀ value of greater2.3 nM.

Example 5: FACs Analysis of Anti-EGFR Antibodies in In VitroKeratinocyte Binding Assay

A keratinocyte FACs binding assay was performed to determine the bindingaffinity of the Ab1 variant antibodies to normal human epidermalkeratinocyte (NHEK) cells. NHEK cells express wild type EGFR.

Cells were harvested when approximately 80% confluent using trypsin,neutralized and washed once in PBS/1% FBS (FACS buffer) then resuspendedat 2.5×10⁶ cells/mL in FACS buffer. 100 μL of cells/well were added to around bottom 96-well plate. 10 μL of a 10× concentration of Ab (finalconcentrations are listed in the Figures) was added and plate wasincubated at 4° C. for one hour. Wells were washed twice with FACSbuffer then resuspended in 50 μL of secondary Ab (AlexaFluor 488)diluted in FACS buffer. The plate was incubated at 4° C. for one hourthen washed twice with FACS buffer. Cells were then resuspended in 100μL of PBS/1% formaldehyde and analyzed on a Becton Dickinson LSRII flowcytometer. Data was analyzed using WinList flow cytometry analysissoftware.

The results from the in vitro keratinocyte binding assay are describedin FIG. 9, which indicates that as the concentration of labeled AbA,AbG, AbK, AbM, and AbP antibody increased, the measured fluorescenceincreased due to binding of EGFR on the keratinocytes. Ab2 was used as apositive control and resulted in increased fluorescence with theaddition of the antibody to the NHEK cells. Ab1 showed much lower levelsof binding, even at high concentrations of antibody.

The results presented in FIG. 9 indicate that the tested Ab1 variantantibodies bind wild type EGFR on keratinocytes and have an affinity tonormal human epidermal keratinocytes which is greater than Ab1 (andnegative control, Control 1 antibody). The results in FIG. 9 also showthat antibodies AbA, AbG, AbK, AbM, and AbP have lower binding to normalhuman epidermal keratinocytes as compared to Ab2. These results indicatethat the Ab1 variant antibodies are able to bind wild type EGFR onkeratinocytes better than Ab1.

Example 6: In Vivo Analysis of Anti-EGFR Antibodies on Tumors

The effect of the Ab1 variant antibodies on the growth of tumors in vivowas evaluated using a mouse xenograft assay.

SCID and athymic CD-1 nude mice were obtained from Charles River(Wilmington, Mass.). Ten mice were housed per cage. The body weight ofthe mice upon arrival was 18-20 g. All experiments were conducted incompliance with the National Institutes of Health Guide for Care and Useof Laboratory Animals guidelines in a facility accredited by theAssociation for the Assessment and Accreditation of Laboratory AnimalCare. For each subcutaneous study, viable cells were inoculatedsubcutaneously into the right flank of the mice on Day 0. The injectionvolume was 0.2 mL composed of a 1:1 mixture of S-MEM and Matrigel (BD,Franklin Lakes, N.J.). Tumors were size matched at approximately 200-250mm³. Therapy began the day of, or 24 hours, after size matching thetumors. A human IgG mixture control was used as a negative control(purified human IgG analogous to human serum; Innovative Research). Miceweighed approximately 25 g at the onset of therapy. Tumor volume wasestimated two to three times weekly. Measurements of the length (L) andwidth (W) of the tumor were taken via electronic caliper and the volumewas calculated according to the following equation: V=(L×W²)/2. Micewere euthanized when tumor volume reached 3,000 mm³ or when skinulcerations occurred. Appropriate amounts of the antibody stock werediluted in phosphate buffered saline prior to administration. Drugs wereadministered intraperitoneally as indicated in the figures. H292 (humannon-small cell lung carcinoma (NSCLC)) cells were used in the xenograftstudy.

Results from the in vivo experiment show that Ab1 variant antibodiesAbA, AbG, AbK, AbM, and AbP were able to significantly inhibit tumorgrowth relative to Ab1 (see FIG. 10). The Ab1 variant antibodies werealso able to increase the durability of the duration of the responsecompared to Ab1, e.g., AbA maintained a tumor volume of less than 500mm³ for 29 days as versus Ab1 which maintained a tumor volume of lessthan 500 mm³ for about 18 days post tumor cell injection (following thesame dose and administration schedule). As shown in FIG. 10, at Day 20,mice injected with AbA showed an approximate tumor volume of 300 mm³,whereas at Day 20 mice injected with Ab1 showed a tumor having anapproximate volume of 700 mm³. By Day 29, AbA injected mice showed atumor having a similar volume as compared to the size at Day 20 (i.e.,approximately 300 mm³), whereas Ab1 injected mice had an increase intumor volume to about 1000 mm³.

The percent tumor growth inhibition (% TGI) was calculated in order toquantitate the results described in FIG. 10. The % TGI for theantibodies described in FIG. 10 are as follows:

-   -   AbA=74    -   AbG=88    -   AbK=90    -   AbM=84    -   AbP=86    -   Ab1=31    -   Ab2=73        The % TGI values are relative to the tumor volumes of mice        treated with the human IgG control. As described above, Ab1        resulted in 31% TGI relative to the human IgG control, whereas        AbA had a calculated 74% TGI. Notably, the Ab1 variant        antibodies showed equal or greater % TGI relative to Ab2.

AbA was able to increase the durability of the response and decreasetumor volume in vivo in a H292 xenograft tumor model (as described inFIG. 10), yet failed to inhibit phosphorylation of EGFR in vitro in thesame cell line, as described in Example 4 and FIG. 7. Antibody AbAactivity in vivo was also comparable to antibodies AbG, AbK, AbM, andAbP despite having a lower binding affinity for EGFR(1-525) and EGFRvIII(see FIG. 3). Thus, despite the fact that AbA showed little to no cellsignaling inhibition in vitro, AbA decreased or inhibited H292 tumorcell growth in vivo in a manner similar to other anti-EGFR variantantibodies having stronger affinity values, e.g., AbK, AbM, and AbP.

Example 7: In Vivo Tumor Growth Inhibition Assay Using AbA ADCs

The ability of an AbA-vcMMAE ADC to inhibit tumor growth was determinedusing an in vivo mouse xenograft assay. The AbA ADC used in this examplewas conjugated according to the method described in Example 8, but wasnot purified according to the batch purification method describedtherein. The average DAR for the AbA ADC composition used in thisexample was 3.7. Mouse xenograft assays (similar to those described inExample 6) were performed using two different NSCLC cells lines,NCI-H1703 and EBC1. Results from the two different cell lines areprovided in FIG. 14. As shown in FIG. 14A, AbA-vcMMAE (at a dose of 1mg/kg body weight) was better at decreasing tumor volume and increasingthe durability of the response in NCI-H1703 cells than Ab1 (at a dose of10 mg/kg body weight) and compared to an Ab1 ADC containing Ab1 and MMAFwith an mc linker. Ab1 alone (at a dose of 10 mg/kg) resulted in anoverall lack of tumor volume inhibition similar to the negative control,antibody control 2 (an anti-tetanus toxin antibody) which did notinhibit tumor growth at a dose of 10 mg/kg.

Efficacy of the AbA-vcMMAE ADC was also studied in a xenograft mousemodel using EBC1 cells. The results from the study are shown in FIG. 14Band indicate that the AbA-mcMMAF ADC (at a dose of 3 mg/kg body weight)was more effective at decreasing tumor volume and increasing thedurability of the response compared to Ab1 (at a dose of 3 mg/kg bodyweight) and compared to an Ab1 ADC containing Ab1 and MMAF with a mclinker (at a dose of 3 mg/kg body weight).

In sum, the results in FIG. 14 show that two different AbA auristatinADCs (AbA-vcMMAE and AbA-mcMMAF) were effective at decreasing tumorvolume and increasing the durability of the response in vivo relative toAb1 alone or an Ab1-MMAF ADC.

Example 8: Purified Anti-EGFR Antibody Drug Conjugates (ADCs)

An antibody drug conjugate (ADC) was made comprising the AbA antibodylinked to monomethyl auristatin E (MMAE) via a valine-citrulline (vc)linker. A diagram of this ADC, referred to as AbA-vcMMAE, is describedin FIG. 11.

Conjugation of the AbA antibody with vcMMAE began with a partialreduction of AbA followed by reaction with Val-Cit-MMAE (vcMMAE). TheAbA antibody (20 mg/mL) was partially reduced by addition of TCEP (molarequivalents of TCEP:mAb is 2.1) followed by incubation at 0° C.overnight. The reduction reaction was then warmed to 20° C. To conjugateall of the thiols, vcMMAE was added to a final vcMMAE:reduced Cys molarratio of 1.15. The conjugation reaction was carried out in the presenceof 10% v/v of DMSO and allowed to proceed at 20° C. for 60 minutes.

After the conjugation reaction, excess free N(acetyl)-Cysteine (2equivalents vs. vcMMAE charge) was added to quench unreacted vcMMAE toproduce the Cys-Val-Cit-MMAE adduct. The Cys quenching reaction wasallowed to proceed at 20° C. for approximately 30 minutes. TheCys-quenched reaction mixture was purified as per below.

The above conjugation method can also be used to conjugate mcMMAF to anantibody.

Batch Purification

The AbA ADCs were purified using a batch purification method. Thereaction mixture was treated with the appropriate amount of water washedBu-HIC resin (ToyoPearl; Tosoh Biosciences), i.e., seven weights ofresin was added to the mixture. The resin/reaction mixture was stirredfor the appropriate time, and monitored by analytical hydrophobicinteraction chromatography for removal of drug conjugate products,filtered through a coarse polypropylene filter, and washed by two bedvolumes of a buffer (0.28 M sodium chloride, 7 mM potassium phosphate,pH 7). The combined filtrate and rinses were combined and analyzed forproduct profile by HIC HPLC. The combined filtrate and rinses werebuffer exchanged by ultrafiltration/diafiltration (UF/DF) to 15 mMhistidine, pH 6 with 10 diavolumes 15 nM histidine buffer.

Following conjugation and purification, analytical analysis of theresulting ADC mixture was performed. Samples were taken and analyzedusing hydrophobic interaction chromatography-high-performance liquidchromatography (HIC-HPLC). The column used was a TSK gel Butyl-NPRcolumn (4.6 mm ID×3.5 cm, 2.5 μm, 30° C.; Tosoh Bioscience LLC, Japan)and was used at a flow rate of 0.8 mL/min. The mobile phases included A:25 mM Na₂HPO₄, pH7, 1.5 M(NH₄)₂SO and B: 25 mM Na₂HPO₄, pH7 (75%) to IPA(25%). The gradient used was 0% phase B for 2 minutes, 0 to 100% B in 12minutes, and hold for 1 minute.

The protein content was analyzed using UV analysis. HIC trace analysisshowed that the resulting average Drug to Antibody Ratio (DAR) forAbA-vcMMAE was 3.1, as described below in Table 4 and FIG. 12. Theaverage DAR was determined by summing up the 0, 1, 2, 3, 4, 5, 6, 7 and8 ADC product, multiplying PA % (PA % is the peak area percent asdetermined by the area measured under the peak at A₂₈₀ by requisite drugload), and dividing by 100.

TABLE 4 Results of HIC Analysis (PA %) of AbA-vcMMAE using BatchPurification HIC pa % results @ 280 nm Rentention Broad Purified timemin DAR Distribution (Batch) 6.7 0 4.27 6.15 7.6 1 0.67 1.43 8.6 2 24.7434.27 9.6 3 1.95 3.94 10.4 4 35.28 45.88 11.5 5 4.67 5.37 11.9 6 13.902.09 12.4 7 6.40 0.88 12.9 8 8.12 Σ (>E6), pa % 280 nm = 28.4 3.0 E4/E2= 1.4 1.3 DAR = 4.1 3.1As can be seen from Table 4 and FIG. 12, batch purification ofAbA-vcMMAE resulted in a DAR of between 2-4. The initial average DAR was4.1, where the final average DAR following purification was 3.1.

The AbA-vcMMAE ADC mixture was also analyzed by size-exclusionchromatography (SEC). SEC HPLC was performed using a Tosoh TSKgelGS3000SWXL column (7.58×30 cm, 5 μm. A flow rate of 0.3 mL/min was used.The mobile phases included 92.5% at 25 mM Na₂PO₄, pH 6.8, 350 mM NaCland 7.5% isopropyl alcohol (IPA). The diluent was the mobile phase, andthe analysis was performed at a UV at 214 nm. SEC pa % results @214 nmare provided below in Table 5. SEC results are shown in FIG. 13.

TABLE 5 SEC Results (PA %) of AbA-vcMMAE High Molecular Low MolecularWeight Monomer Weight Broad 3.2 96.8 Purified 2.2 97.8 DAR 2 0.4 99.6DAR4 1.0 96.2 2.8 mAb 95.9 AbA SEC PA % results at 214 nmThus, batch purification of AbA-vcMMAE resulted in a DAR of the between2-4, with an average of 3.1.

Example 9: In Vivo Tumor Growth Inhibition Assay Using AbA-MMAE ADCs

As described in Example 8, purified compositions of AbA-vcMMAE wereprepared such that the average DAR of the ADCs within the compositionwas 3.1. The purified ADCs were subsequently tested to determine whetherthe purified AbA ADC composition was effective at inhibiting tumorgrowth in vivo using a lung cancer xenograft model. More specifically,xenograft tumor growth inhibition assays were performed to assess theeffect of purified AbA-vcMMAE (Ab1-vcMMAEp) on NCI-H292 cells (a humanNSCLC carcinoma cell line).

The results provided in FIG. 15A demonstrate that AbA-vcMMAEp (at a doseof 3 mg/kg body weight) was more effective at decreasing tumor volumeand extending the duration of the response (increasing the durability ofthe response) in NCI-H292 cells when compared to the unpurifiedAbA-vcMMAE. Similarly, FIG. 15B demonstrates that the purifiedAbA-vcMMAEp (at a dose of 6 mg/kg body weight) was also more effectiveat inhibiting tumor growth when compared to the unpurified form ofAbA-vcMMAE. As described in both FIGS. 15A and 15B, purified AbA-vcMMAEwas also more effective at decreasing tumor volume and extending theduration of the response than the negative Control 2 antibody (alone orconjugated to MMAE), Ab1 conjugated to MMAE or MMAF in either theunpurified or purified form. The control 2 antibody is an anti-tetanustoxin antibody which does not bind to EGFR.

In a further study assessing the effect of purified AbA-vcMMAE(Ab1-vcMMAEp) on NCI-H292 cells (lung carcinoma cell line; ATCC®CRL-1848™), purified AbA-vcMMAE was tested at a dose of 3 mg/kg and 6mg/kg versus Ab1-mcMMAF at similar doses. The results of this secondstudy in the NSCLC tumor model are provided in FIG. 18, and demonstratethat purified AbA-vcMMAEp was more effective at inhibiting tumor growthwhen compared to AbA-vcMMAE.

Example 10: Flow Through Process Purification of AbA-vcMMAE

Compositions comprising AbA-vcMMAE ADCs with reduced drug loads ofvc-MMAE molecules per antibody were made using the flow through process.Preparation of the AbA-vcMMAE ADCs is described above in Example 7.

A flow through process for purifying a composition of AbA-vcMMAE havingADCs with a range of DARs (1-8) was performed according to thefollowing. A 5 mL column of the Bu-HIC resin was first equilibrated withapproximately 28 mM NaCl, 7 mM potassium phosphate, pH 7. The reactionmixture was subsequently diluted with ⅙ its volume using 1.95 M NaCl, 50mM potassium phosphate, pH 7, and loaded into the resin at anapproximate ratio of 100 mg of protein/mL of resin at a flow rate of 1mL/min (approximately 5 min residence time or 36 cm/hr linear flow). Arinse consisting of 1 part isopropanol and 10 parts (by volume) of 28 mMNaCl, 7 mM potassium phosphate, pH 7, was applied as a rinse for about12 column volumes. The product was collected beginning approximatelyafter 1 column volume, until after the UV signal equilibrated (which mayalso be collected in fractions). The fractions were analyzed by HIC HPLCand the desired aliquots were pooled together and concentrated by TFF(tangential flow filtration) and exchanged to 15 mM histidine, pH 6 with10 diavolumes of the histidine buffer. Table 6 below, provide purityresults for the reaction mixture before and after purification.

TABLE 6 Results of HIC Analysis (PA %) of AbA-vcMMAE using Flow ThroughPurification HIC PA % results @ 280 nm Broad Purified Rt, min DARDistribution (Flow Through) 6.4 0 3.89 5.25 7.2 1 0.67 0.99 8.4 2 22.3630.21 9.4 3 2.95 4.04 10.2 4 39.82 52.76 11.3 5 5.41 4.67 11.8 6 12.521.42 12.3 7 5.31 0.67 12.9 8 7.07 Σ (>E6), pa % 280 nm = 24.9 2.1 E4/E2= 1.8 1.7 DAR = 4.1 3.2

In comparison to the batch purification described in Example 8, loadingof protein versus resin was comparable for each purification mode (batchpurification and flow through purification). In the batch purificationprocess, 2.26 weights of resin was used versus ADC on a potency adjustedbasis. The density of the resin was approximately 0.23 g/ml. Thus, forexample, if 10 g ADC was used, 98.2 ml resin was used. The loading usedwas (10 g×1000 ml/L)/98.2 ml=102 g/L loading. The flow throughpurification experiments target a loading of 100 g/L. In each case theload solution and rinse solutions were the same, except that the flowthrough purification rinse solution was 10% IPA v/v.

A comparison between a purified AbA-vcMMAE composition obtained usingthe batch purification method described in Example 8 and a purifiedAbA-vcMMAE composition obtained using the flow through process describedabove, is provided in Table 7.

TABLE 7 Results of HIC Analysis (PA %) of AbA-vcMMAE Batch Purificationversus Flow Through Purification HIC PA % results @ 280 nm PurifiedPurified Rt, min DAR (Batch) (Flow Through) 6.4 0 4.99 5.25 7.2 1 1.020.99 8.4 2 30.99 30.21 9.4 3 4.61 4.04 10.2 4 50.75 52.76 11.3 5 5.54.67 11.8 6 2.1 1.42 12.3 7 — 0.67 Σ (>E6), pa % 280 nm = 2.1 2.1 E4/E2= 1.6 1.7 DAR = 3.2 3.2

In sum, both the flow through and batch purification processes weresuccessful in obtaining a composition comprising about 80% ADCs having aDAR of 2-4, where the amount of ADCs having a DAR of 0-1 or 5-8 waslimited to less than about 20% of the overall ADC population in thecomposition, as shown, e.g., in Table 7.

Example 11: In Vivo Tumor Growth Inhibition Assay Using AbA MMAE ADCs

The ability of an AbA-vcMMAE ADC to inhibit glioblastoma tumor growthwas determined using an in vivo mouse xenograft assay. Mouse xenograftassays (similar to those performed in Example 6) were performed usingU87MGde2-7 cells (which express EGFRvIII). U87 cells are derived from ahuman maliganant gliomas (ATCC No. HTB-14™). For the study, tumor cellswere mixed with 50% Matrigel (BD BioSciences, Franklin Lakes, N.J.) and3×10⁶ cells were inoculated subcutaneously into the flank of 6-8 weekold Nu/Nu female mice at Day 0 (Nu/Nu female mice were obtained fromCharles River (Wilmington, Mass.)). Measurements of the length (L) andwidth (W) of the tumor were taken via electronic caliper and the volumewas calculated according to the following equation: V=(L×W²)/2. Micewere euthanized when tumor volume reached 3,000 mm³ or when skinulcerations occurred. The results provided in FIG. 19 demonstrate thatAbA-vcMMAEp was more effective than the negative control (Control 2antibody; anti-tetanus antibody) in inhibiting glioblastoma cell growth.

SEQUENCE SUMMARY

SEQ ID NO: Description 1 Ab1 VH amino acid sequence 2 Ab1, AbC, AbD, andAbE VH CDR1 amino acid sequence 3 Ab1, AbC, AbD, AbE, AbF, AbJ, and AbNVH CDR2 amino acid sequence 4 Ab1, AbC, AbD, and AbE VH CDR3 amino acidsequence 5 Ab1 and AbA VL amino acid sequence 6 Ab1, AbA, AbB, AbC, andAbF VL CDR1 amino acid sequence 7 Ab1, AbA, AbB, and AbC, and AbF VLCDR2 amino acid sequence 8 Ab1, AbA, AbB, and AbF VL CDR3 amino acidsequence 9 AbA VH amino acid sequence 10 AbA, AbF, and AbK VH CDR1 aminoacid sequence 11 AbA, AbH, AbK, AbL, AbM, AbO, and AbQ VH CDR2 aminoacid sequence 12 AbA, AbF, AbM, AbN, and AbO VH CDR3 amino acid sequence13 Ab1 and AbA light chain amino acid sequence 14 Ab1 heavy chain aminoacid sequence 15 AbA heavy chain amino acid sequence 16 AbB and AbG VHCDR1 amino acid sequence 17 AbB and AbG VH CDR2 amino acid sequence 18AbG, AbH, AbJ, and AbL VH CDR3 amino acid sequence 19 AbB and AbK VHCDR3 amino acid sequence 20 AbM and AbN VH CDR1 amino acid sequence 21AbP VH CDR1 amino acid sequence 22 AbP and AbQ VH CDR3 amino acidsequence 23 AbG, AbH, and AbJ VL CDR1 amino acid sequence 24 AbG, AbH,and AbJ VL CDR2 amino acid sequence 25 AbG, AbH, and AbJ VL CDR3 aminoacid sequence 26 AbK, AbL, AbM, AbN, and AbO VL CDR1 amino acid sequence27 AbE, AbK, AbL, AbM, AbN, and AbO VL CDR2 amino acid sequence 28 AbK,AbL, AbM, AbN, and AbO VL CDR3amino acid sequence 29 AbP and AbQ VL CDR1amino acid sequence 30 AbP and AbQ VL CDR2 amino acid sequence 31 AbD,AbP, and AbQ VL CDR3 amino acid sequence 32 Human EGFR amino acidsequence (with signal sequence) 33 Human Epidermal Growth FactorReceptor variant III (hEGFRvIII) amino acid sequence 34 Human EGFRextracellular domain (ECD) amino acid sequence 35 VH CDR1 consensussequence of AbA, AbG, AbK, AbM, and AbP 36 VH CDR2 consensus sequence ofAbA, AbG, AbK, AbM, and AbP 37 VH CDR3 consensus sequence of AbA, AbG,AbK, AbM, and AbP 38 VL CDR1 consensus sequence of AbA, AbG, AbK, AbM,and AbP 39 VL CDR2 consensus sequence of AbA, AbG, AbK, AbM, and AbP 40VL CDR3 consensus sequence of AbA, AbG, AbK, AbM, and AbP 41 Ig gamma-1constant region 42 Ig gamma-1 constant region mutant 43 Ig kappaconstant region 44 Ig lambda constant region 45 Epitope of EGFR 46 ECDof EGFRvIII amino acid sequence 47 EGFR 1-525 amino acid sequence 48Heavy chain amino acid sequence Ab2 49 Light chain amino acid sequenceAb2 50 VH amino acid sequence AbE 51 VL amino acid sequence AbE 52 VHamino acid sequence AbF 53 VL amino acid sequence AbF 54 VH amino acidsequence AbH 55 VL amino acid sequence AbH 56 VH amino acid sequence AbJ57 VL amino acid sequence AbJ 58 VH amino acid sequence AbL 59 VL aminoacid sequence AbL 60 VH amino acid sequence AbN 61 VL amino acidsequence AbN 62 VH amino acid sequence AbO 63 VL amino acid sequence AbO64 VH amino acid sequence AbB 65 VL amino acid sequence AbB 66 VH aminoacid sequence AbC 67 VL amino acid sequence AbC 68 VH amino acidsequence AbD 69 VL amino acid sequence AbD 70 VH amino acid sequence AbQ71 VL amino acid sequence AbQ 72 VH amino acid sequence AbG 73 VL aminoacid sequence AbG 74 VH amino acid sequence AbK 75 VL amino acidsequence AbK 76 VH amino acid sequence AbM 77 VL amino acid sequence AbM78 VH amino acid sequence AbP 79 VL amino acid sequence AbP 80 AbH, AbJ,AbL, and AbO VH CDR1 amino acid sequence 81 AbQ VH CDR1 amino acidsequence 82 AbD and AbE VL CDR1 amino acid sequence 83 AbD VL CDR2 aminoacid sequence 84 AbC VL CDR3 amino acid sequence 85 AbE VL CDR3 aminoacid sequence 86 AbA heavy chain nucleic acid sequence 87 AbA lightchain nucleic acid sequence 88 Heavy chain amino acid leader sequence 89Light chain amino acid leader sequence

INCORPORATION BY REFERENCE

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An anti-human epidermal growth factor receptor (anti-hEGFR) antibodythat a) binds to an epitope within the amino acid sequenceCGADSYEMEEDGVRKC (SEQ ID NO: 45) or competes with a second anti-hEGFRantibody for binding to epidermal growth factor receptor variant III(EGFRvIII) (SEQ ID NO: 33) in a competitive binding assay, wherein thesecond anti-EGFR antibody comprises a heavy chain variable domaincomprising the amino acid sequence set forth in SEQ ID NO: 1 and a lightchain variable domain comprising the amino acid sequence set forth inSEQ ID NO: 5; b) binds to EGFR(1-525) (SEQ ID NO: 47) with adissociation constant (K_(d)) of about 1×10⁻⁶ M or less, as determinedby surface plasmon resonance; and c) inhibits tumor growth in an in vivohuman non-small-cell lung carcinoma (NSCLC) xenograft assay with a tumorgrowth inhibition % (TGI %) of at least about 50% relative to a humanIgG antibody which is not specific for EGFR, wherein the human IgGantibody is administered in the NSCLC xenograft assay at the same doseand frequency as the anti-hEGFR antibody.
 2. The antibody of claim 1,which binds to EGFR (1-525) (SEQ ID NO: 47) with a K_(d) of betweenabout 1×10⁻⁶ M and about 1×10⁻¹⁰ M, as determined by surface plasmonresonance.
 3. The antibody of claim 1, which binds to EGFR (1-525) (SEQID NO: 47) with a K_(d) of between about 1×10⁻⁶ M and about 1×10⁻⁷ M, asdetermined by surface plasmon resonance.
 4. The antibody of claim 1,which binds to EGFRvIII (SEQ ID NO: 33) with a K_(d) of about 8.2×10⁻⁹ Mor less, as determined by surface plasmon resonance.
 5. The antibody ofclaim 1, which binds to EGFRvIII (SEQ ID NO: 33) with a K_(d) of betweenabout 8.2×10⁻⁹ M and about 6.3×10⁻¹⁰ M, as determined by surface plasmonresonance.
 6. The antibody of claim 1, which binds to EGFRvIII (SEQ IDNO: 33) with a K_(d) of between about 8.2×10⁻⁹ M and about 2.0×10⁻⁹ M,as determined by surface plasmon resonance.
 7. The antibody of claim 1,which inhibits tumor growth by at least about 60% in an in vivo humannon-small-cell lung carcinoma (NSCLC) xenograft assay relative to ahuman IgG antibody which is not specific for EGFR.
 8. The antibody ofclaim 1, which inhibits tumor growth by at least about 70% in an in vivohuman non-small-cell lung carcinoma (NSCLC) xenograft assay relative toa human IgG antibody which is not specific for EGFR.
 9. The antibody ofclaim 1, which inhibits tumor growth by at least about 80% in an in vivohuman non-small-cell lung carcinoma (NSCLC) xenograft assay relative toa human IgG antibody which is not specific for EGFR.
 10. A method fortreating a subject having cancer, comprising administering an effectiveamount of an antibody drug conjugate (ADC) comprising an anti-EGFRantibody conjugated to at least one auristatin, wherein the anti-EGFRantibody is an IgG isotype; comprises a heavy chain variable regioncomprising a CDR3 domain comprising the amino acid sequence set forth inSEQ ID NO: 12, a CDR2 domain comprising the amino acid sequence setforth in SEQ ID NO: 11, and a CDR1 domain comprising the amino acidsequence set forth in SEQ ID NO: 10; and comprises a light chainvariable region comprising a CDR3 domain comprising the amino acidsequence set forth in SEQ ID NO: 8, a CDR2 domain comprising the aminoacid sequence set forth in SEQ ID NO: 7, and a CDR1 domain comprisingthe amino acid sequence set forth in SEQ ID NO:
 6. 11. The method ofclaim 10, wherein the antibody comprises a heavy chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO: 9, and alight chain variable region comprising the amino acid sequence set forthin SEQ ID NO:
 5. 12. The method of claim 10, wherein the antibodycomprises a heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 15, and a light chain comprising the amino acid sequence setforth in SEQ ID NO:
 13. 13. The method of claim 10, wherein the canceris selected from the group consisting of breast cancer, lung cancer, aglioblastoma, prostate cancer, pancreatic cancer, colon cancer,colorectal cancer, head and neck cancer, mesothelioma, kidney cancer,squamous cell carcinoma, triple negative breast cancer, and non-smallcell lung cancer.
 14. The method of claim 13, wherein the cancer ischaracterized as having EGFR overexpression.
 15. The method of claim 10,wherein the auristatin is monomethylauristatin E (MMAE).
 16. The methodof claim 15, wherein the antibody is conjugated to MMAE via amaleimidocaproyl, valine-citrulline linker.
 17. The method of claim 16,wherein the ADC is administered in combination with an additional agentor an additional therapy.
 18. The method of claim 17, wherein theadditional agent is selected from the group consisting of an anti-PD1antibody, an anti-CTLA-4 antibody, temozolomide, a bcl-xl inhibitor,ibrutinib, duvelisib, idelalisib, venetoclax, and a nicotinamidephosphoribosyltransferase (NAMPT) inhibitor.
 19. The method of claim 17,wherein the additional therapy is radiation.
 20. The method of claim 17,wherein the additional agent is a chemotherapeutic agent.